Hppd variants and methods of use

ABSTRACT

In the present invention, HPPD polypeptides and plants containing them showing a full tolerance against one or more HPPD inhibitor herbicides belonging to various chemical classes are described. A set of mutant HPPD polypeptides have been designed which have either no or only a significantly reduced affinity to HPPD inhibitor herbicides and, at the same time, the rate of dissociation of the HPPD inhibitors of the mutant HPPD polypeptide is increased to such an extent that the HPPD inhibitors no longer act as slow-binding or slow, tight-binding inhibitors but, instead of this, have become fully reversible inhibitors. In particular, isolated polynucleotides encoding mutant HPPD polypeptides conferring tolerance to HPPD inhibitor herbicides belonging to various chemical classes are provided. Additionally, amino acid sequences corresponding to the polynucleotides are encompassed.

FIELD OF THE INVENTION

This invention relates to plant molecular biology, particularly novelHPPD polypeptides that confer improved tolerance to HPPD inhibitorherbicides.

BACKGROUND OF THE INVENTION

The 4-hydroxyphenylpyruvate dioxygenases (HPPDs) are enzymes whichcatalyze the reaction in which para-hydroxyphenylpyruvate (abbreviatedherein as HPP), a tyrosine degradation product, is transformed intohomogentisate (abbreviated herein as HGA), the precursor in plants oftocopherol and plastoquinone (Crouch N. P. et al. (1997), Tetrahedron,53, 20, 6993-7010, Fritze et al. (2004), Plant Physiology134:1388-1400). Tocopherol acts as a membrane-associated antioxidant.Plastoquinone, firstly acts as an electron carrier between PSII and thecytochrome b6/f complex and secondly, is a redox cofactor for phytoenedesaturase, which is involved in the biosynthesis of carotenoids.

Up to now, more than 1000 nucleic acid sequences from various organismspresent in the NCBI database were annotated as coding for a putativeprotein having an HPPD domain. But for most of those, it has not beenproven that the protein would have an HPPD enzymatic activity, neitherin an in vitro assay, nor in an in planta approach, nor that such HPPDprotein can confer herbicide tolerance to HPPD inhibitor herbicides whenexpressed in a plant. Several HPPD proteins and their primary sequenceshave been described in the state of the art, in particular the HPPDproteins of bacteria such as Pseudomonas (Rüetschi et al., Eur. J.Biochem., 205, 459-466, 1992, WO96/38567), Kordia (WO2011/076889)Synechococcus (WO2011/076877), Acidobacterium and Mucilaginibacter(WO2015/022634), Rhodococcus (WO2011/076892), of protists such asBlepharisma (WO2011/076882), of euryarchaeota such as Picrophilus(WO2011/076885), of algae such as Chlamydomonas reinhardtii (ES2275365;WO2011145015), Scenedesmus (WO2015/022634), of plants such asArabidopsis (WO96/38567, GENBANK® AF047834), carrot (WO 96/38567,GENBANK® 87257), Avena sativa (WO2002/046387, WO2011/068567), wheat(WO2002/046387), Brachiaria platyphylla (WO2002/046387), Cenchrusechinatus (WO2002/046387), Lolium rigidum (WO2002/046387), Festucaarundinacea (WO2002/046387), Setaria faberi (WO 2002/046387), Eleusineindica (WO2002/046387), Sorghum (WO2002/046387, WO2012/021785), corn(WO2012/021785), Coptis japonica (WO2006/132270), Lemna (WO2015/022634),or of mammals such as mouse or pig, or of fungi such as Coccicoides(GENBANK® COITRP).

Inhibition of HPPD leads to uncoupling of photosynthesis, deficiency inaccessory light-harvesting pigments and, most importantly, todestruction of chlorophyll by UV-radiation and reactive oxygen species(bleaching) due to the lack of photo-protection normally provided bycarotenoids (Norris et al. (1995), Plant Cell 7: 2139-2149). Bleachingof photosynthetically active tissues leads to growth inhibition andplant death.

Some molecules which inhibit HPPD (hereinafter named HPPD inhibitorherbicides), and which inhibit transformation of the HPP into HGA whilebinding specifically to the enzyme, have proven to be very effectiveherbicides.

At present, most commercially available HPPD inhibitor herbicides belongto one of these chemical families, as listed below:

1) the triketones, e.g. benzobicyclon [i.e.3-[2-chloro-4-(methylsulfonyl)benzoyl]-4-(phenylsulfanyl)bicyclo[3.2.1]oct-3-en-2-one];sulcotrione [i.e.2-[2-chloro-4-(methylsulfonyl)benzoyl]-1,3-cyclohexanedione], mesotrione[i.e.2-[4-(methylsulfonyl)-2-nitrobenzoyl]-1,3-cyclohexanedione](abbreviatedherein as MST); tembotrione [i.e.2-[2-chloro-4-(methylsulfonyl)-3-[(2,2,2,-trifluoroethoxy)methyl]benzoyl]-1,3-cyclohexanedione];tefuryltrione [i.e.2-[2-chloro-4-(methylsulfonyl)-3-[[(tetrahydro-2-furanyl)methoxy]methyl]benzoyl]-1,3-cyclohexanedione]];bicyclopyrone [i.e.4-hydroxy-3-[[2-[(2-methoxyethoxy)methyl]-6-(trifluoromethyl)-3-pyridinyl]carbonyl]bicyclo[3.2.1]oct-3-en-2-one];fenquinotrione [i.e.2-[[8-chloro-3,4-dihydro-4-(4-methoxyphenyl)-3-oxo-2-quinoxalinyl]carbonyl]-1,3-cyclohexanedione],and as described in WO2007/088876, WO2009/016841, WO2010/089993,WO2010/116122, WO2012/002096, WO2011/31658, WO2012/136703, JP2013040141,WO2013/080484, WO2014/014904, WO2014/031971, US2014/0106968;2) the diketonitriles, e.g.2-cyano-3-cyclopropyl-1-(2-methylsulphonyl-4-trifluoromethylphenyl)-propane-1,3-dioneand2-cyano-1-[4-(methylsulphonyl)-2-trifluoromethylphenyl]-3-(1-methylcyclopropyl)propane-1,3-dione;3) the isoxazoles, e.g. isoxaflutole [i.e.(5-cyclopropyl-4-isoxazolyl)[2-(methylsulfonyl)-4-(trifluoromethyl)phenyl]methanone].In plants, isoxaflutole (abbreviated herein as IFT) is rapidlymetabolized to DKN, a diketonitrile compound which exhibits the HPPDinhibitor property;4) the hydroxypyrazoles, e.g. pyrazoxyfen [i.e.2-[[4-(2,4-dichlorobenzoyl)-1,3-dimethyl-1H-pyrazol-5-yl]oxy]-1-phenylethanone];benzofenap [i.e.2-[[4-(2,4-dichloro-3-methylbenzoyl)-1,3-dimethyl-1H-pyrazol-5-yl]oxy]-1-(4-methylphenyl)ethanone];pyrazolynate [i.e.(2,4-dichlorophenyl)[1,3-dimethyl-5-[[(4-methylphenyl)sulfonyl]oxy]-1H-pyrazol-4yl]methanone];pyrasulfotole [i.e.(5-hydroxy-1,3-dimethyl-1H-pyrazol-4-yl)[2-(methylsulfonyl)-4-(trifluoromethyl)phenyl]methanone];topramezone [i.e.[3-(4,5-dihydro-3-isoxazolyl)-2-methyl-4-(methylsulfonyl)phenyl](5-hydroxy-1-methyl-1H-pyrazol-4-yl)methanone]; tolpyralate [i.e.1-[[1-ethyl-4-[3-(2-methoxyethoxy)-2-methyl-4-(methylsulfonyl)benzoyl]-1H-pyrazol-5-yl]oxy]ethyl methyl carbonate];5) N-(1,2,5-oxadiazol-3-yl)benzamides as described in WO2011/035874, andWO2012/123416, WO2012/123409, EP2562174, WO2013/064459, WO2013/087577,WO2013/124238, WO2013/124228, WO2013/164333, WO2013/037342,WO2014/053473, WO2014/086737, WO2015/007662, WO2015/007632,WO2015/007633, and as described in WO2013/072300, WO2013/072402,WO2013/072450, WO2014/184014, WO2014/184019, WO2014/184058,WO2014/192936, WO2015/052152, WO2015/052178 and theN-(1,3,4-oxadiazol-2-yl)benzamides as described in WO2012/126932, andEP2562174, WO2013/064459, WO2013/087577, WO2013/124238, WO2013/124228,WO2013/124245, WO2013/164333, WO2013/037342, WO2014/1053473,WO2014/086737, WO2015/007662, WO2015/007632, WO2015/007633; e.g.2-methyl-N-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide;2-chloro-N-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide;2-chloro-3-(ethylsulfonyl)-N-(5-methyl-1,3,4-oxadiazol-2-yl)-4-(trifluoromethyl)benzamide;6) N-(tetrazol-5-yl)- or N-(triazol-5-yl)arylcarboxamides as describedin WO2012/028579, and WO2012/123409, WO2013/017559, EP2562174,WO2013/064459, WO2013/064457, WO2013/087577, WO2013/104705,WO2013/124238, WO2013/124228, WO2013/124245, WO2013/164331,WO2013/164333, WO2013/174843, WO2013/037342, WO2014/053473,WO2014/086737, WO2015/007662, WO2015/007632, WO2015/007633; e.g.2-chloro-3-ethoxy-4-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide,4-(difluoromethyl)-2-methoxy-3-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide,2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide(hereinafter also named “Cmpd. 1”),2-(methoxymethyl)-3-(methylsulfinyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide,and as described in WO2013/072528, WO2013/076315, WO2013/076316,WO2013/083859, WO2013/092834, WO2013/139760, WO2013/144231,WO2014/126070, WO2014/135654, WO2014/184015, WO2014/184016,WO2014/184017, WO2014/184073, WO2014/184074, WO2014/192936,WO2015/022284, WO2015/052153, WO2015/052173;7) pyridazinone derivatives as described in WO2013/050421 andWO2013/083774, WO2014/154828, WO2014/154882;8) oxoprazine derivatives as described in WO2013/054495;9) N-(triazol-2-yl)arylcarboxamides as described in WO2013/144234,WO2015/007564;10) triazinones as described in WO2014/154829; and11) pyrazolones as described in EP2881387 and EP2881388.

These HPPD inhibitor herbicides can be used against grass and/or broadleaf weeds in field of crop plants that display metabolic tolerance,such as maize (Zea mays), rice (Oryza sativa) and wheat (Triticumaestivum) in which they are rapidly degraded (Schulz et al. (1993), FEBSletters, 318, 162-166; Mitchell et al. (2001), Pest Management Science,Vol 57, 120-128; Garcia et al. (2000), Biochem., 39, 7501-7507; Pallettet al. (2001), Pest Management Science, Vol 57, 133-142). In order toextend the scope of use of these HPPD inhibitor herbicides, severalefforts have been developed in order to confer to plants, particularlyplants without or with an underperforming metabolic tolerance, atolerance level acceptable under agronomic field conditions.

Besides the attempt of by-passing HPPD-mediated production ofhomogentisate (U.S. Pat. No. 6,812,010), overexpressing the sensitiveenzyme so as to produce quantities of the target enzyme in the plantwhich are sufficient in relation to the herbicide has been performed(WO96/38567). Overexpression of HPPD polypepdtides resulted in betterpre-emergence tolerance to the diketonitrile derivative (abbreviatedherein as DKN) of IFT, but the tolerance level was not sufficient fortolerance to post-emergence treatment (Matringe et al. (2005), PestManagement Science 61: 269-276).

A third strategy was to mutate the HPPD polypeptide in order to obtain atarget enzyme which, while retaining its properties of catalyzing thetransformation of HPP into HGA, is less sensitive to HPPD inhibitorsthan is the native HPPD polypeptide before mutation.

This strategy has been successfully applied for the production of plantstolerant to2-cyano-3-cyclopropyl-1-(2-methylsulphonyl-4-trifluoromethylphenyl)-propane-1,3-dioneand to2-cyano-1-[4-(methylsulphonyl)-2-trifluoromethylphenyl]-3-(1-methylcyclopropyl)propane-1,3-dione(EP496630), two HPPD inhibitor herbicides belonging to thediketonitriles family (WO99/24585). Pro215Leu, Gly336Glu, Gly336Ile, andmore particularly Gly336Trp (positions of the mutated amino acid areindicated with reference to the wild-type Pseudomonas fluorescens HPPDpolypeptide corresponding to SEQ ID NO: 1 of present invention) wereidentified as mutations which are responsible for an increased toleranceto treatment with these diketonitrile herbicides.

Quite recently, introduction of a Pseudomonas fluorescens HPPD gene intothe plastid genome of tobacco and soybean has shown to be more effectivethan nuclear transformation, conferring tolerance to post-emergenceapplication of IFT (Dufourmantel et al. (2007), Plant Biotechnol J.5(1):118-33).

In WO2004/024928, the inventors sought to increase the prenylquinonebiosynthesis (e.g. synthesis of plastoquinones, tocopherols) in thecells of plants by increasing the flux of the HPP precursor into thecells of these plants. This has been done by connecting the synthesis ofsaid precursor to the “shikimate” pathway by overexpression of aprephenate dehydrogenase (PDH) enzyme. They have also noted that thetransformation of plants with a gene encoding a PDH enzyme and a geneencoding an HPPD enzyme makes it possible to increase the tolerance ofsaid plants to HPPD inhibitor herbicides.

In WO2009/144079, nucleic acid sequences encoding anhydroxyphenylpyruvate dioxygenase (HPPD) with specific mutations atposition 336 of the Pseudomonas fluorescens HPPD protein and their usefor obtaining plants which are tolerant to HPPD inhibitor herbicides wasdisclosed.

In WO2002/046387, several domains of HPPD polypeptides originating fromplants have been identified that may be relevant to confer tolerance tovarious HPPD inhibitor herbicides but neither in planta nor biochemicaldata have been shown to confirm the impact of the as described domainfunctions.

In WO2008/150473, the combination of two distinct tolerance mechanisms—amodified Avena sativa gene coding for a mutant HPPD enzyme and a CYP450Maize monooxygenase (nsfl gene)—was exemplified in order to obtain animproved tolerance to HPPD inhibitor herbicides, but no data have beendisclosed demonstrating the synergistic effects based on the combinationof both proteins.

Further, a method to generate plants tolerant to HPPD inhibitorherbicides by overexpressing not only a gene coding for a tolerant HPPD,as for example from Avena sativa (US2011/0173718) or Arabidopsis(WO2013/064964, WO2014/177999), but also in combination with severalplant genes coding for an HST (homogentisate solanesyltransferase)protein is disclosed. However, the level of tolerance to some selectedHPPD inhibitor herbicides was rather limited.

In WO2011/094199 and US2011/0185444, the tolerance of several hundred ofsoybean wild-type lines to the HPPD inhibitor IFT was evaluated. Veryfew lines displayed reasonable level of tolerance to the herbicides. Theputative QTL (quantitative trait loci) responsible for the tolerance wasidentified. In this region of the genome, a gene coding for an ABCtransporter was identified as being the main trait responsible for theimproved tolerance to the HPPD inhibitor herbicide observed. However,transgenic plants expressing the identified genes did not display anyimprovement in tolerance to the tested HPPD inhibitor herbicides.

In WO2010/085705 and US2014/0053295, several mutants of the Avena sativaHPPD polypeptide were disclosed. In WO2010/085705 it was shown that someof the variants displayed improved tolerance in vitro to the triketone“Mesotrione” (abbreviated herein as MST), however, only very few mutantswere expressed in tobacco plants. Additionally, none of the tobaccoplants expressing these mutants displayed improved tolerance to MST orIFT compared to tobacco plants expressing the wild-type Avena sativaHPPD gene. In US2014/0053295, a few Avena sativa HPPD mutants wereexpressed in soybean plants and had good tolerance level to MST as knownfrom plants expressing the wild-type Avena sativa HPPD gene. However,other herbicides such as tembotrione or IFT induced much higher leafdamage in these soybean plants.

US 2012/0042413 describes mutant maize HPPD polypeptides having HPPDactivity but also showing a certain insensitivity to at least one HPPDinhibitor herbicide and further suggests a certain set of mutations atdifferent positions of HPPD polypeptides and finally disclosesbiochemical data as well as tolerance levels of plants containing few ofsuch mutated HPPD polypeptides. In EP 2453012, several mutants of HPPDpolypeptides have been described; however, the improved tolerance of thedescribed mutants was not demonstrated in planta against several HPPDinhibitor herbicides.

In WO2014/043435, recombinant nucleic acid molecules encoding thePseudomonas spp. HPPD polypeptides consisting of an amino acid sequencecomprising a proline at the amino acid position corresponding to aminoacid position 335 of SEQ ID NO: 1; or a proline at the amino acidposition corresponding to amino acid position 335 of SEQ ID NO:1 and atryptophan at the amino acid position corresponding to amino acidposition 336 of SEQ ID NO: 1; or a serine at the amino acid positioncorresponding to amino acid position 335 of SEQ ID NO: 1, a serine atthe amino acid position corresponding to amino acid position 336 of SEQID NO:1, a threonine at the amino acid position corresponding to aminoacid position 339 of SEQ ID NO:1, and a glutamine at the amino acidposition corresponding to amino acid position 340 of SEQ ID NO: 1; or atryptophan at the amino acid position corresponding to amino acidposition 188 of SEQ ID NO:1 and a tryptophan at the amino acid positioncorresponding to amino acid position 336 of SEQ ID NO: 1; or a prolineat the amino acid position corresponding to amino acid position 335 ofSEQ ID NO: 1, a serine at the amino acid position corresponding to aminoacid position 336 of SEQ ID NO: 1, and a glutamic acid at the amino acidposition corresponding to amino acid position 340 of SEQ ID NO: 1; or aproline at the amino acid position corresponding to amino acid position335 of SEQ ID NO:1, a tryptophan at the amino acid positioncorresponding to amino acid position 336 of SEQ ID NO: 1, an alanine atthe amino acid position corresponding to amino acid position 339 of SEQID NO:1, and a glutamine at the amino acid position corresponding toamino acid position 340 of SEQ ID NO:1 were described.

In WO2015/135881, recombinant nucleic acid molecules encoding thePseudomonas spp. HPPD polypeptides consisting of an amino acid sequencecomprising a proline at the amino acid position corresponding to aminoacid position 335 of SEQ ID NO: 1, and a phenylalanine or tyrosine atthe amino acid position corresponding to amino acid position 336 of SEQID NO: 1; and, optionally, one or more additional substitutions at theamino acid positions corresponding to amino acid position 188, 189, 200,215, 226, 339, and 340 of SEQ ID NO:1, were described.

The currently described and partly commercialized HPPD inhibitorherbicides act as slow-binding or slow, tight-binding inhibitors (seeMorrison (1982) Trends Biochem. Sci. 7, 102-105). These inhibitors bindslowly (i.e. they have slow rates of association, kon) but notcovalently to the HPPD polypeptide (i.e. they produce time-dependentinhibition), and are released very slowly (i.e. they have exceptionallyslow rates of dissociation, koff) due to their exceedingly tightinteraction with the enzyme. These inhibitors bind so tightly thatstoichiometric titrations with the enzyme are possible.

It has become increasingly recognized that the slow-binding or slow,tight-binding inhibitors are not only extraordinary potentHPPD-inhibitor, but, in addition, have features that make themattractive agrochemicals for weed control. The slow rate of dissociationenhances inhibitor effectiveness to such an extent that ideally only oneinhibitor molecule per HPPD polypeptide active site is sufficient tofully inhibit the activity and to maintain this level of inhibition fora long time period even in the absence of free inhibitor molecules inthe plant cell. This translates into low application rates of theseinhibitors to control undesired weeds in crop growing areas.

The properties of slow-binding or slow, tight-binding inhibitors areadvantageous when achieving HPPD inhibition and herbicidal activity isthe goal. However, these properties are a major disadvantage when HPPDpolypeptides tolerant to these inhibitors are to be invented. Mutationsin the HPPD polypeptide that solely reduce the affinity of the inhibitorto the enzyme (ki) do not fully overcome HPPD inhibition since bindingof the inhibitor and inhibition of the HPPD polypeptide can still takeplace and, therefore, the achieved level of inhibition will bemaintained for a long time period even in the absence of free inhibitorin the plant cell. In addition the in part commercially available HPPDinhibitor herbicides belong to structurally diverse chemical classes,such as the triketones, the diketonitriles, the isoxazoles, thehydroxypyrazoles, the N-(1,2,5-oxadiazol-3-yl)benzamides, theN-(1,3,4-oxadiazol-2-yl)benzamides, the N-(tetrazol-5-yl)- orN-(triazol-5-yl)arylcarboxamides, the pyridazinone derivatives, theoxoprazine derivatives, the N-(triazol-2-yl), the triazinones, and thepyrazolones. The currently described state of the art HPPD polypeptidesdemonstrate a rather narrow range of tolerance to structurally diverseHPPD inhibitor herbicides.

Due to the above described kinetic properties of all the currentlydescribed and partly commercialized HPPD inhibitor herbicides, up tonow, no HPPD inhibitor herbicide tolerant plants with full toleranceagainst HPPD inhibitor herbicides have been published, despite the manyefforts to generate them.

SUMMARY OF INVENTION

In the present invention, HPPD polypeptides and plants containing them,showing a full tolerance against one or more HPPD inhibitor herbicidesbelonging to various chemical classes, are described. It turned out thatin order to generate such HPPD polypeptides with maximized and broadtolerance against several classes of HPPD inhibitor herbicides, it isimportant to reduce the affinity to the HPPD polypeptide (ki) concerningthe respective HPPD inhibitor herbicide(s) and simultaneously to ensurean improved rate of dissociation (koff) of a slow-binding or slow,tight-binding inhibitor as known from the wild-type and several mutantHPPD polypeptides to achieve high level of inhibitor tolerance.

In the present invention, this goal was achieved by developing a set ofHPPD polypeptides, which have either no or only a significantly reducedaffinity to HPPD inhibitor herbicides and, at the same time, the rate ofdissociation of the HPPD inhibitor herbicides of the enzyme is increasedto such an extent that the HPPD inhibitor herbicides no longer act asslow-binding or slow, tight-binding inhibitors but, instead of this,have become fully reversible inhibitors.

In the present invention, compositions and methods for obtaining a newset of HPPD polypeptides having the before mentioned characteristics(i.e. no or only a significantly reduced affinity to HPPD inhibitorherbicides, increased rate of dissociation of the HPPD inhibitorherbicides of the enzyme; HPPD inhibitor herbicides no longer act asslow-binding or slow, tight-binding inhibitors but have become fullyreversible inhibitors) are provided. Compositions include HPPDpolypeptides and isolated, recombinant or chimeric nucleic acidmolecules encoding such HPPD polypeptides, vectors and host cellscomprising those nucleic acid molecules. Compositions also include theantibodies to those polypeptides. The nucleotide sequences can be usedin DNA constructs or expression cassettes for transformation andexpression in organisms, including microorganisms and plants. Thenucleotide sequences may be synthetic sequences that have been designedfor expression in an organism including, but not limited to, amicroorganism or a plant.

The compositions include nucleic acid molecules encoding herbicidetolerant HPPD polypeptides, including nucleic acid molecules encoding anHPPD polypeptide having (a) an alanine at the amino acid positioncorresponding to amino acid position 268 of SEQ ID NO:1, (b) a prolineat the amino acid position corresponding to amino acid position 335 ofSEQ ID NO:1, (c) a histidine or an aspartic acid at the positioncorresponding to amino acid position 336 of SEQ ID NO:1, and (d) aserine at the position corresponding to amino acid position 337 of SEQID NO: 1, and wherein said HPPD polypeptide is tolerant to one or moreHPPD inhibitor herbicide(s) and, optionally, one or more further aminoacid substitutions at the positions corresponding to amino acidpositions 213, 215, 270, 315, 340, 344, 345 of SEQ ID NO: 1, includingthe HPPD polypeptides set forth in any of SEQ ID NO:2 to SEQ ID NO:71 aswell as fragments thereof.

Compositions also comprise transformed plants, plant cells, tissues, andseeds that are tolerant to the HPPD inhibitor herbicides by theintroduction of the nucleic acid sequence of the invention into thegenome of the plants, plant cells, tissues, and seeds. The introductionof the sequence allows for HPPD inhibitor herbicides to be applied toplants to selectively kill HPPD inhibitor sensitive weeds or otheruntransformed plants, but not the transformed organism. The sequencescan additionally be used as a marker for selection of plant cellsgrowing in the presence of one or more HPPD inhibitor herbicides.

Methods for identifying HPPD polypeptides with HPPD inhibitor herbicidetolerance activity are additionally provided.

The compositions and methods of the invention are useful for theproduction of organisms with enhanced tolerance to HPPD inhibitorherbicides. These organisms and compositions comprising the organismsare desirable for agricultural purposes. Plants or seeds comprising thenucleic acid sequence encoding an HPPD polypeptide according to theinvention can be grown in a field and harvested to obtain a plantproduct. The compositions of the invention are also useful for detectingthe presence of HPPD inhibitor herbicide tolerant polypeptides ornucleic acids in products or organisms.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a simplistic scheme of the coupled HPPD activity assay usedin this invention to determine the enzymatic activity of the exemplaryHPPD polypeptides.

FIG. 2 shows exemplary kinetic changes in absorbance at 320 nm (Abs320)in raw extracts samples of wild-type and knock-out HPPD polypeptideobserved with 200 μM HPP and 0, 4 or 13 μM Cmpd. 1(2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide)according to Example 3 in the coupled HPPD activity assay. The knock-outHPPD polypeptide was obtained by exchanging a histidine to an alanine atthe amino acid position corresponding to amino acid position 162 of SEQID NO:1. This position is well known for its importance due to itsinvolvement in the coordinated binding of the iron atom in the activesite of the HPPD polypeptide (Serre et al. (1999), Structure, 7,977-988).

DETAILED DESCRIPTION OF THE INVENTION

The present inventions now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the inventions are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

Overview

Several efforts have been developed in order to confer to plants anagronomically-acceptable level of tolerance to a broad range of HPPDinhibitor herbicides, including by-passing HPPD-mediated production ofhomogentisate (U.S. Pat. No. 6,812,010), overexpressing the sensitiveenzyme so as to produce quantities of the target enzyme in the plant,which are sufficient in relation to the herbicide (WO96/38567), andmutating the HPPD in order to obtain a target enzyme which, whileretaining its properties of catalyzing the transformation of HPP intohomogentisate, is less sensitive to HPPD inhibitors than is the nativeHPPD before mutation.

Despite these successes obtained for the development of plants showingtolerance to several HPPD inhibitor herbicides described above, it isstill necessary to develop and/or improve the tolerance of plants tonewer or to several different HPPD inhibitor herbicides belonging tovarious chemical classes, particularly HPPD inhibitor herbicidesbelonging to the classes of triketones (e.g. benzobicyclon, sulcotrionemesotrione, tembotrione, tefuryltrione, bicyclopyrone, fenquinotrione),diketonitriles, isoxazoles (e.g. isoxaflutole), hydroxypyrazoles (e.g.pyrazoxyfen, benzofenap, pyrazolynate, pyrasulfotole, topramezone,tolpyralate), N-(1,2,5-oxadiazol-3-yl)benzamides,N-(1,3,4-oxadiazol-2-yl)benzamides (e.g.2-methyl-N-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide,N-(tetrazol-5-yl)- or N-(triazol-5-yl)arylcarboxamides (e.g.2-chloro-3-ethoxy-4-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide),4-(difluoromethyl)-2-methoxy-3-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide),2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide(hereinafter also named “Cmpd. 1”),2-(methoxymethyl)-3-(methylsulfinyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide,pyridazinone derivatives, oxoprazine derivatives, triketones,N-(triazol-2-yl)arylcarboxamides, triazinones, and pyrazolones.

Thus, the present invention provides improved compositions and methodsfor regulating HPPD inhibitor herbicide tolerance. HPPD inhibitorherbicides like those of the class of triketones (e.g. benzobicyclon,sulcotrione mesotrione, tembotrione, tefuryltrione, bicyclopyrone,fenquinotrione), diketonitriles, isoxazoles (e.g. isoxaflutole),hydroxypyrazoles (e.g. pyrazoxyfen, benzofenap, pyrazolynate,pyrasulfotole, topramezone, tolpyralate),N-(1,2,5-oxadiazol-3-yl)benzamides, N-(1,3,4-oxadiazol-2-yl)benzamides(e.g.2-methyl-N-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide,N-(tetrazol-5-yl)- or N-(triazol-5-yl)arylcarboxamides (e.g.2-chloro-3-ethoxy-4-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide,4-(difluoromethyl)-2-methoxy-3-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide,2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide(hereinafter also named “Cmpd. 1”),2-(methoxymethyl)-3-(methylsulfinyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide,pyridazinone derivatives, oxoprazine derivatives,N-(triazol-2-yl)arylcarboxamides, triazinones, and pyrazolones have anoutstanding herbicidal activity against a broad spectrum of economicallyimportant monocotyledonous and dicotyledonous annual harmful plants. Theactive substances also act efficiently on perennial harmful plants,which produce shoots from rhizomes, wood stocks or other perennialorgans and which are difficult to control. Within the meaning of thepresent invention, “herbicide” is understood as being a herbicidallyactive substance on its own or such a substance which is combined withan additive which alters its efficacy, such as, for example, an agentwhich increases its activity (a synergistic agent) or which limits itsactivity (a safener). The herbicide may further comprise solid or liquidadjuvants or carriers that are ordinarily employed in formulationtechnology (e.g. natural or regenerated mineral substances, solvents,dispersants, wetting agents, tackifiers, emulsifiers, growth promotingagents, and the like), as well as one or more additional herbicidesand/or one or more pesticides (e.g. insecticides, virucides,microbicides, amoebicides, pesticides, fungicides, bactericides,nematicides, molluscicides, and the like).

The methods involve transforming organisms with nucleotide sequencesencoding an HPPD inhibitor herbicide tolerance gene of the invention orotherwise introducing such HPPD inhibitor herbicide tolerance genes inorganisms not containing them (e.g. by mating, cell fusion, or bycrossing organisms containing an introduced HPPD inhibitor herbicidetolerance gene of the invention with organisms not containing it andobtaining progeny containing such gene). The nucleotide sequences of theinvention are useful for preparing plants that show increased toleranceto HPPD inhibitor herbicides, particularly increased tolerance to HPPDinhibitor herbicides of the class of triketones (preferablybenzobicyclon, sulcotrione, mesotrione, tembotrione, tefuryltrione,bicyclopyrone, or fenquinotrione), diketonitriles, isoxazoles(preferably isoxaflutole), hydroxypyrazoles (preferably pyrazoxyfen,benzofenap, pyrazolynate, pyrasulfotole, topramezone, or tolpyralate),N-(1,2,5-oxadiazol-3-yl)benzamides, N-(1,3,4-oxadiazol-2-yl)benzamides(preferably2-methyl-N-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide,N-(tetrazol-5-yl)- or N-(triazol-5-yl)arylcarboxamides (preferably2-chloro-3-ethoxy-4-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide,4-(difluoromethyl)-2-methoxy-3-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide,2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide(hereinafter also named “Cmpd. 1”),2-(methoxymethyl)-3-(methylsulfinyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide,pyridazinone derivatives, oxoprazine derivatives,N-(triazol-2-yl)arylcarboxamides, triazinones, and pyrazolones.

The expression of the HPPD inhibitor herbicide tolerance gene of theinvention may also result in tolerance towards the “coumarone-derivativeherbicides” (described in WO2009/090401, WO2009/090402, WO2008/071918,WO2008/009908). In this regard, any one of the HPPD inhibitor herbicidetolerance genes of the invention can also be expressed in a plant alsoexpressing a chimeric homogentisate solanesyltransferase (HST) gene or amutated HST gene as described in WO2011/145015, WO2013/064987,WO2013/064964, or WO2010/029311, to obtain plants tolerant to HSTinhibitor herbicides. As used herein, a “coumarone-derivative herbicide”or “HST inhibitor herbicide” encompasses compounds which fall under theIUPAC nomenclature of 5H-thiopyrano[4,3-b]pyridin-8-ol,5H-thiopyrano[3,4-b]pyrazin-8-ol, oxathiino[5,6-b]pyridin-4-ol, andoxathiino[5, 6-b]pyrazin-4-ol.

Thus, by “HPPD inhibitor herbicide tolerance” gene of the invention isintended a gene encoding a polypeptide that confers upon a cell ororganism the ability to tolerate a higher concentration of an HPPDinhibitor herbicide than such cell or organism that does not express theprotein, or to tolerate a certain concentration of an HPPD inhibitorherbicide for a longer time than such cell or organism that does notexpress the protein, or that confers upon a cell or organism the abilityto perform photosynthesis, grow, and/or reproduce with less damage orgrowth inhibition observed than such cell or organism not expressingsuch protein.

An “HPPD inhibitor herbicide tolerance polypeptide” comprises apolypeptide that confers upon a cell or organism the ability to toleratea higher concentration of HPPD inhibitor herbicides than such cell ororganism that does not express the protein, or to tolerate a certainconcentration of HPPD inhibitor herbicides for a longer period of timethan such cell or organism that does not express the polypeptide, orthat confers upon a cell or organism the ability to performphotosynthesis, grow, and/or reproduce with less damage or growthinhibition observed than such cell or organism not expressing suchpolypeptide.

The term “polypeptide” comprises proteins such as enzymes, antibodiesand medium-length polypeptides and short peptides down to an amino acidsequence length below ten.

The term “enzyme” means in the present invention any polypeptidecatalyzing the reaction in which para-hydroxyphenylpyruvate istransformed into homogentisate. It includes naturally-occurring enzymes,as well as enzyme variants and derivatives thereof. It also comprisesany fragment of such an enzyme, and variants engineered by insertion,deletion, recombination and/or any other method, that leads to enzymesthat differ in their amino acid sequence from the naturally-occurringenzyme or the enzyme variants. It also comprises protein molecules withposttranslational and/or chemical modifications, e.g. glycosylation,gamma carboxylation and acetylation, any molecular complex or fusionprotein comprising one of the aforementioned proteins.

The terms “polypeptide variant” or “mutant polypeptide” means anypolypeptide molecule obtained by mutagenesis, preferably bysite-directed or random mutagenesis with an altered amino acid sequencecompared to the respective wild-type sequence. By “tolerate”,“tolerance” or “resistant” is intended either to survive a particularHPPD inhibitor herbicide application, or the ability to carry outessential cellular functions such as photosynthesis, protein synthesisor respiration and reproduction in a manner that is not readilydiscernable from untreated cells or organisms, or the ability to have nosignificant difference in yield or even improved yield for plantstreated with HPPD inhibitor herbicide compared to such plants nottreated with such herbicide (but where weeds have been removed orprevented by a mechanism other than application of the HPPD inhibitorherbicide, such as the methods described in WO2011/100302, which isherein incorporated by reference in its entirety).

In addition to conferring upon a cell HPPD inhibitor herbicidetolerance, the HPPD nucleic acid sequences of the invention encodepolypeptides having HPPD activity, i.e. catalyzing the reaction in whichpara-hydroxyphenylpyruvate (HPP) is transformed into homogentisate. Thecatalytic activity of an HPPD polypeptide may be defined by variousmethods well-known in the art. WO2009/144079 and WO2014/043435 describevarious suitable screening methods.

The enzymatic activity of HPPD polypeptides can be measured by anymethod that makes it possible either to measure the decrease in theamount of the HPP or O₂ substrates, or to measure the accumulation ofany of the products derived from the enzymatic reaction, i.e.homogentisate or CO₂. In particular, the HPPD activity can be measuredby means of the method described in WO2009/144079; Garcia et al. (1997),Biochem. J. 325, 761-769; Garcia et al. (1999), Plant Physiol. 119,1507-1516; or in WO2012/021785, which are incorporated herein byreference.

For the purposes of the present invention, a “reference” HPPDpolypeptide (or HPPD gene encoding such polypeptide) is any HPPDpolypeptide or nucleic acid against which the HPPD polypeptide or HPPDnucleic acid of the invention is being compared. For the purposes ofdescribing the HPPD polypeptides of the present invention, the terms“protein” and “polypeptide” are used interchangeably.

This reference HPPD polypeptide can be a native plant, bacterial, oranimal HPPD, or can be a mutated HPPD polypeptide that is known in theart such as the PfP215L and PfG336F mutants described in WO2009/144079,or can be either of the PfHPPDevo33, PfHPPDevo36, PfHPPDevo37,PfHPPDevo40, or PfHPPDevo41 proteins of WO2014/043435; PfHPPDevo41 isset forth in present application as SEQ ID NO:2. Such reference HPPDpolypeptide can be used to determine whether the HPPD polypeptide ornucleic acid of the invention has a particular property of interest(e.g., improved, comparable or decreased HPPD inhibitor herbicidetolerance or HPPD polypeptide enzymatic activity; improved, comparableor decreased expression in a host cell; improved, comparable ordecreased protein stability, and the like).

In various embodiments herein, the HPPD inhibitor herbicide tolerantpolypeptide encoded by a nucleic acid (including isolated, recombinantand chimeric genes thereof, vectors, host cells, plants, plant parts,and seeds comprising the nucleic acid, HPPD polypeptides andcompositions thereof encoded by the nucleic acid, as well as methods ofusing the polypeptide encoded by the nucleic acid for increasingtolerance of a plant to HPPD inhibitor herbicides, particularlyincreased tolerance to HPPD inhibitor herbicides of the class oftriketones (preferably benzobicyclon, sulcotrione, mesotrione,tembotrione, tefuryltrione, bicyclopyrone, fenquinotrione),diketonitriles, isoxazoles (preferably isoxaflutole), hydroxypyrazoles(preferably pyrazoxyfen, benzofenap, pyrazolynate, pyrasulfotole,topramezone, tolpyralate), N-(1,2,5-oxadiazol-3-yl)benzamides,N-(1,3,4-oxadiazol-2-yl)benzamides (preferably2-methyl-N-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide,N-(tetrazol-5-yl)- or N-(triazol-5-yl)arylcarboxamides (preferably2-chloro-3-ethoxy-4-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide,4-(difluoromethyl)-2-methoxy-3-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide,2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide(hereinafter also named “Cmpd. 1”),2-(methoxymethyl)-3-(methylsulfinyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide),pyridazinone derivatives, oxoprazine derivatives,N-(triazol-2-yl)arylcarboxamides, triazinones, and pyrazolones) has (a)an alanine at the amino acid position corresponding to amino acidposition 268 of SEQ ID NO:1, (b) a proline at the amino acid positioncorresponding to amino acid position 335 of SEQ ID NO:1, (c) a histidineor an aspartic acid at the position corresponding to amino acid position336 of SEQ ID NO:1, and (d) a serine at the position corresponding toamino acid position 337 of SEQ ID NO: 1, and wherein said HPPDpolypeptide is tolerant to one or more HPPD inhibitor herbicide(s) and,optionally, one or more further amino acid substitutions at thepositions corresponding to amino acid positions 213, 215, 270, 315, 340,344, 345 of SEQ ID NO: 1, including the HPPD polypeptides set forth inany of SEQ ID NO:2 to NO:71. By “corresponding to” is intended thenucleotide or amino acid position relative to that position in SEQ IDNO:1 when two (or more) sequences are aligned using standard alignmentalgorithms. The term “position” in a polynucleotide or polypeptiderefers to specific single bases or amino acids in the sequence of thepolynucleotide or polypeptide, respectively. The term “site” in apolynucleotide or polypeptide refers to a certain position or region inthe sequence of the polynucleotide or polypeptide, respectively. Theterm “polynucleotide” corresponds to any genetic material of any lengthand any sequence, comprising single-stranded and double-stranded DNA andRNA molecules, including regulatory elements, structural genes, groupsof genes, plasmids, whole genomes, and fragments thereof.

In one embodiment, the HPPD polypeptide of the present invention(including the nucleotide sequence encoding it and recombinant andchimeric genes thereof, vectors, host cells, plants, plant parts, andseeds comprising the nucleotide sequence encoding the HPPD polypeptideof the invention) consists of an amino acid sequence comprising

(a) an alanine at the amino acid position corresponding to amino acidposition 268 of SEQ ID NO: 1, (b) a proline at the amino acid positioncorresponding to amino acid position 335 of SEQ ID NO:1, (c) a histidineor an aspartic acid at the position corresponding to amino acid position336 of SEQ ID NO: 1, and (d) a serine at the position corresponding toamino acid position 337 of SEQ ID NO:1, and wherein said HPPDpolypeptide is tolerant to one or more HPPD inhibitor herbicide(s).

In another embodiment, the HPPD polypeptide of the present invention(including the nucleotide sequence encoding it and recombinant andchimeric genes thereof, vectors, host cells, plants, plant parts, andseeds comprising the nucleotide sequence encoding the HPPD polypeptideof the invention) being tolerant to one or more HPPD inhibitorherbicides consists of an amino acid sequence comprising (a) an alanineat the amino acid position corresponding to amino acid position 268 ofSEQ ID NO: 1, (b) a proline at the amino acid position corresponding toamino acid position 335 of SEQ ID NO: 1, (c) a histidine or an asparticacid at the position corresponding to amino acid position 336 of SEQ IDNO: 1, and (d) a serine at the position corresponding to amino acidposition 337 of SEQ ID NO:1, and further comprising

-   -   i. a lysine or leucine at the amino acid position corresponding        to amino acid position 213 of SEQ ID NO:1; and/or    -   ii. an alanine at the amino acid position corresponding to amino        acid position 215 of SEQ ID NO:1; and/or    -   iii. an arginine, asparagine, leucine, glutamic acid, proline or        serine at the amino acid position corresponding to amino acid        position 270 of SEQ ID NO: 1; and/or    -   iv. an arginine, lysine, glutamine, methionine or histidine at        the amino acid position corresponding to amino acid position 315        of SEQ ID NO:1; and/or    -   v. an arginine, glutamine, methionine, glutamic acid, glycine,        leucine, or valine at the amino acid position corresponding to        amino acid position 340 of SEQ ID NO: 1; and/or    -   vi. a glutamine, proline, or arginine at the amino acid position        corresponding to amino acid position 344 of SEQ ID NO:1; and/or    -   vii. a lysine, arginine, methionine, alanine, or valine at the        amino acid position corresponding to amino acid position 345 of        SEQ ID NO:1.

In another embodiment, the HPPD polypeptide of the present invention(including the nucleotide sequence encoding it and recombinant andchimeric genes thereof, vectors, host cells, plants, plant parts, andseeds comprising the nucleotide sequence encoding the HPPD polypeptideof the invention) being tolerant to one or more HPPD inhibitorherbicides consists of an amino acid sequence comprising

(a) an alanine at the amino acid position corresponding to amino acidposition 268 of SEQ ID NO: 1, (b) a proline at the amino acid positioncorresponding to amino acid position 335 of SEQ ID NO:1, (c) a histidineor an aspartic acid at the position corresponding to amino acid position336 of SEQ ID NO: 1, and (d) a serine at the position corresponding toamino acid position 337 of SEQ ID NO:1, and further comprising

-   -   i. a leucine or lysine at the amino acid position corresponding        to amino acid position 213 of SEQ ID NO:1; and/or    -   ii. an alanine at the amino acid position corresponding to amino        acid position 215 of SEQ ID NO:1; and/or    -   iii. an asparagine, glutamic acid or serine at the amino acid        position corresponding to amino acid position 270 of SEQ ID NO:        1; and/or    -   iv. an arginine, lysine, glutamine or methionine at the amino        acid position corresponding to amino acid position 315 of SEQ ID        NO:1; and/or    -   v. an arginine, or valine at the amino acid position        corresponding to amino acid position 340 of SEQ ID NO:1; and/or    -   vi. a glutamine at the amino acid position corresponding to        amino acid position 344 of SEQ ID NO:1; and/or    -   vii. a lysine, valine, or methionine at the amino acid position        corresponding to amino acid position 345 of SEQ ID NO:1.

In another embodiment, the HPPD polypeptide of the present invention(including the nucleotide sequence encoding it and recombinant andchimeric genes thereof, vectors, host cells, plants, plant parts, andseeds comprising the nucleotide sequence encoding the HPPD polypeptideof the invention) being tolerant to one or more HPPD inhibitorherbicides consists of an amino acid sequence comprising

(a) an alanine at the amino acid position corresponding to amino acidposition 268 of SEQ ID NO: 1, (b) a proline at the amino acid positioncorresponding to amino acid position 335 of SEQ ID NO:1, (c) a histidineor an aspartic acid at the position corresponding to amino acid position336 of SEQ ID NO: 1, and (d) a serine at the position corresponding toamino acid position 337 of SEQ ID NO:1, and further comprising

-   -   i. a lysine at the amino acid position corresponding to amino        acid position 213 of SEQ ID NO:1; and/or    -   ii. an alanine at the amino acid position corresponding to amino        acid position 215 of SEQ ID NO:1; and/or    -   iii. an asparagine, glutamic acid or serine at the amino acid        position corresponding to amino acid position 270 of SEQ ID NO:        1; and/or    -   iv. an arginine, lysine, glutamine at the amino acid position        corresponding to amino acid position 315 of SEQ ID NO:1; and/or    -   v. an arginine or valine at the amino acid position        corresponding to amino acid position 340 of SEQ ID NO:1; and/or    -   vi. a glutamine at the amino acid position corresponding to        amino acid position 344 of SEQ ID NO:1; and/or    -   vii. a lysine, valine, or methionine at the amino acid position        corresponding to amino acid position 345 of SEQ ID NO:1.

In another embodiment, the HPPD polypeptide of the present invention(including the nucleotide sequence encoding it and recombinant andchimeric genes thereof, vectors, host cells, plants, plant parts, andseeds comprising the nucleotide sequence encoding the HPPD polypeptideof the invention) being tolerant to one or more HPPD inhibitorherbicides consists of an amino acid sequence comprising

(a) an alanine at the amino acid position corresponding to amino acidposition 268 of SEQ ID NO: 1, (b) a proline at the amino acid positioncorresponding to amino acid position 335 of SEQ ID NO: 1, (c) ahistidine at the position corresponding to amino acid position 336 ofSEQ ID NO: 1, and (d) a serine at the position corresponding to aminoacid position 337 of SEQ ID NO: 1, and further comprising

-   -   i. a lysine at the amino acid position corresponding to amino        acid position 213 of SEQ ID NO: 1, and/or    -   ii. an asparagine, glutamic acid or serine at the amino acid        position corresponding to amino acid position 270 of SEQ ID        NO:1, and/or    -   iii. a arginine, lysine, glutamine at the amino acid position        corresponding to amino acid position 315 of SEQ ID NO:1; and/or    -   iv. a valine at the amino acid position corresponding to amino        acid position 340 of SEQ ID NO: 1; and/or    -   v. a glutamine at the amino acid position corresponding to amino        acid position 344 of SEQ ID NO: 1, and/or    -   vi. a valine, lysine, or methionine at the amino acid position        corresponding to amino acid position 345 of SEQ ID NO:1.

In another embodiment, the HPPD polypeptide of the present invention(including the nucleotide sequence encoding it and recombinant andchimeric genes thereof, vectors, host cells, plants, plant parts, andseeds comprising the nucleotide sequence encoding the HPPD polypeptideof the invention) being tolerant to one or more HPPD inhibitorherbicides consists of an amino acid sequence comprising

(a) an alanine at the amino acid position corresponding to amino acidposition 268 of SEQ ID NO: 1, (b) a proline at the amino acid positioncorresponding to amino acid position 335 of SEQ ID NO:1, (c) a histidineor an aspartic acid at the position corresponding to amino acid position336 of SEQ ID NO: 1, and (d) a serine at the position corresponding toamino acid position 337 of SEQ ID NO:1, and further comprising

-   -   i. an alanine at the amino acid position corresponding to amino        acid position 215 of SEQ ID NO:1, and/or    -   ii. asparagine, glutamic acid or serine at the amino acid        position corresponding to amino acid position 270 of SEQ ID        NO:1, and/or    -   iii. an arginine, glutamine at the amino acid position        corresponding to amino acid position 315 of SEQ ID NO:1; and/or    -   iv. a valine at the amino acid position corresponding to amino        acid position 340 of SEQ ID NO:1, and/or    -   v. a glutamine at the amino acid position corresponding to amino        acid position 344 of SEQ ID NO:1; and/or    -   vi. a lysine, valine, or methionine at the amino acid position        corresponding to amino acid position 345 of SEQ ID NO:1.

In another embodiment, the HPPD polypeptide of the present invention(including the nucleotide sequence encoding it and recombinant andchimeric genes thereof, vectors, host cells, plants, plant parts, andseeds comprising the nucleotide sequence encoding the HPPD polypeptideof the invention) being tolerant to one or more HPPD inhibitorherbicides consists of an amino acid sequence comprising

(a) an alanine at the amino acid position corresponding to amino acidposition 268 of SEQ ID NO: 1, (b) a proline at the amino acid positioncorresponding to amino acid position 335 of SEQ ID NO: 1, (c) ahistidine at the position corresponding to amino acid position 336 ofSEQ ID NO: 1, and (d) a serine at the position corresponding to aminoacid position 337 of SEQ ID NO:1, and further comprising

-   -   i. an asparagine or serine at the amino acid position        corresponding to amino acid position 270 of SEQ ID NO:1;    -   ii. an arginine or glutamine at the amino acid position        corresponding to amino acid position 315 of SEQ ID NO:1;    -   iii. a valine at the amino acid position corresponding to amino        acid position 340 of SEQ ID NO:1;    -   iv. a glutamine at the amino acid position corresponding to        amino acid position 344 of SEQ ID NO: 1, and    -   v. a valine, lysine, or methionine at the amino acid position        corresponding to amino acid position 345 of SEQ ID NO:1.

In another embodiment, the HPPD polypeptide of the present invention(including the nucleotide sequence encoding it and recombinant andchimeric genes thereof, vectors, host cells, plants, plant parts, andseeds comprising the nucleotide sequence encoding the HPPD polypeptideof the invention) being tolerant to one or more HPPD inhibitorherbicides consists of an amino acid sequence comprising

(a) an alanine at the amino acid position corresponding to amino acidposition 268 of SEQ ID NO: 1, (b) a proline at the amino acid positioncorresponding to amino acid position 335 of SEQ ID NO:1, (c) an asparticacid at the position corresponding to amino acid position 336 of SEQ IDNO:1, and (d) a serine at the position corresponding to amino acidposition 337 of SEQ ID NO:1, and further comprising

-   -   i. a serine at the amino acid position corresponding to amino        acid position 270 of SEQ ID NO:1,    -   ii. a valine at the amino acid position corresponding to amino        acid position 340 of SEQ ID NO:1;    -   iii. a glutamine at the amino acid position corresponding to        amino acid position 344 of SEQ ID NO: 1, and    -   iv. a methionine at the amino acid position corresponding to        amino acid position 345 of SEQ ID NO:1.

In another embodiment, the HPPD polypeptide of the present invention(including the nucleotide sequence encoding it and recombinant andchimeric genes thereof, vectors, host cells, plants, plant parts, andseeds comprising the nucleotide sequence encoding the HPPD polypeptideof the invention) being tolerant to one or more HPPD inhibitorherbicides consists of an amino acid sequence comprising

(a) an alanine at the amino acid position corresponding to amino acidposition 268 of SEQ ID NO: 1, (b) a proline at the amino acid positioncorresponding to amino acid position 335 of SEQ ID NO: 1, (c) anaspartic acid at the position corresponding to amino acid position 336of SEQ ID NO:1, and (d) a serine at the position corresponding to aminoacid position 337 of SEQ ID NO: 1, and further comprising

-   -   i. a asparagine at the amino acid position corresponding to        amino acid position 270 of SEQ ID NO:1,    -   ii. a valine at the amino acid position corresponding to amino        acid position 340 of SEQ ID NO:1;    -   iii. a glutamine at the amino acid position corresponding to        amino acid position 344 of SEQ ID NO: 1, and    -   iv. a methionine at the amino acid position corresponding to        amino acid position 345 of SEQ ID NO:1.

Table 1 summarizes the respective amino acid positions in comparison tothe reference wild-type Pseudomonas fluorescens HPPD polypeptide (SEQ IDNO:1) where the HPPD polypeptide variants according to the inventioncomprising three or more amino acid substitutions. If not otherwiseexplicitly stated the exchanges at the relevant amino acid positions arealways referred to the reference wild-type Pseudomonas fluorescens HPPDpolypeptide corresponding to SEQ ID NO:1.

TABLE 1 Overview of exemplary amino acid exchanges relative to the HPPDpolypeptide corresponding to SEQ ID NO: 1 Amino acid position relativeto Exemplary amino SEQ ID NO: 1 acid exchanges 213 L, K 215 A 268 A 270R, N, L, E, P, S 315 R, K, Q, M, H 335 P 336 D, H 337 S 340 G, R, E, V,Q, M, L 344 Q, P, R 345 V, K, M, R, A

Amino acids are referred to herein using the name of the amino acid, thethree letter abbreviation or the single letter abbreviation. The tablebelow provides a list of the standard amino acids together with theirabbreviations.

Alanine A Ala Cysteine C Cys Aspartic acid D Asp Glutamic acid E GluPhenylalanine F Phe Glycine G Gly Histidine H His Isoleucine I IleLysine K Lys Leucine L Leu Methionine M Met Asparagine N Asn Proline PPro Glutamine Q Gln Arginine R Arg Serine S Ser Threonine T Thr Valine VVal Tryptophan W Trp Tyrosine Y Tyr Cysteine C Cys

It is well known to one of ordinary skill in the art that the geneticcode is degenerate, that is more than one codon triplet can code for thesame amino acid. Therefore, the amino acid sequences provided herein,can be generated by alternate sequences that use different codons toencode the same amino acid sequence.

In another embodiment, HPPD polypeptides according to the invention haveat least 53%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity to the amino acid sequenceset forth herein as SEQ ID NO:1.

Exemplary HPPD sequences that can be modified according to the presentinvention include those from bacteria, particularly from Pseudomonasspp. type, more particularly from Pseudomonas fluorescens, Pseudomonasputida, Pseudomonas aeruginosa, Pseudomonas testosteroni (Comamonastestosteroni).

For the purposes of the present invention, the HPPD polypeptide of theinvention may also comprise further modifications, for example, whereinsome amino acids (e.g. 1 to 17 amino acids) have been replaced, added ordeleted for cloning purposes, to make a transit peptide fusion, and thelike, which retains HPPD activity, i.e. the property of catalyzing theconversion of para-hydroxyphenylpyruvate to homogentisate, or can be anyHPPD polypeptide that can be further improved. For example, the HPPDpolypeptide that can be further improved by the modifications describedherein can be the variant HPPD derived from Pseudomonas fluorescens setforth herein as any of SEQ ID NO:2 to NO:71.

In a preferred embodiment, HPPD polypeptides according to presentinvention and being tolerant to one or more HPPD inhibitor herbicidesare equivalent to SEQ ID NO:1 (Pseudomonas fluorescens) beside the aminoacids being replaced according to present invention, i.e. the respectiveHPPD polypeptide is identical to SEQ ID NO:1 but having

(a) an alanine at the amino acid position corresponding to amino acidposition 268 of SEQ ID NO: 1, (b) a proline at the amino acid positioncorresponding to amino acid position 335 of SEQ ID NO: 1, (c) ahistidine or an aspartic acid at the position corresponding to aminoacid position 336 of SEQ ID NO:1, and (d) a serine at the positioncorresponding to amino acid position 337 of SEQ ID NO:1.

In a further preferred embodiment, HPPD polypeptides according topresent invention being tolerant to one or more HPPD inhibitorherbicides are equivalent to SEQ ID NO:1 (Pseudomonas fluorescens)beside the amino acids being replaced according to present invention,ie., the respective HPPD polypeptide is identical to SEQ ID NO:1 buthaving one or more amino acid exchanges at the respective amino acidposition according to Table 1, above, with the proviso that an alanineexists at position 268 of SEQ ID NO:1, a proline exists at position 335of SEQ ID NO:1, a histidine or an aspartic acid exists at position 336of SEQ ID NO:1 and a serine exists at position 337 of SEQ ID NO:1.

In a further preferred embodiment, HPPD polypeptides according topresent invention being tolerant to one or more HPPD inhibitorherbicides are equivalent to SEQ ID NO:1 (Pseudomonas fluorescens)beside the amino acids being replaced according to present invention,ie., the respective HPPD polypeptide is identical to SEQ ID NO:1 buthaving amino acid exchanges at respective amino acid position(s) asdefined in Table 2 (below).

In some embodiments, the nucleotide sequence of the invention (includingisolated, recombinant and chimeric genes thereof, vectors, host cells,plants, plant parts, and seeds comprising the nucleic acid sequence,amino acid sequences and compositions thereof encoded by the nucleicacid sequence, as well as methods of using the nucleic acid sequence forincreasing tolerance of a plant to HPPD inhibitor herbicides,particularly increased tolerance to HPPD inhibitor herbicides of theclass of triketones (preferably benzobicyclon, sulcotrione, mesotrione,tembotrione, tefuryltrione, bicyclopyrone, fenquinotrione),diketonitriles, isoxazoles (preferably isoxaflutole) hydroxypyrazoles(preferably pyrazoxyfen, benzofenap, pyrazolynate, pyrasulfotole,topramezone, tolpyralate), N-(1,2,5-oxadiazol-3-yl)benzamides,N-(1,3,4-oxadiazol-2-yl)benzamides (preferably2-methyl-N-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide,N-(tetrazol-5-yl)- or N-(triazol-5-yl)arylcarboxamides (preferably2-chloro-3-ethoxy-4-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide,4-(difluoromethyl)-2-methoxy-3-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide,2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide(hereinafter also named “Cmpd. 1”),2-(methoxymethyl)-3-(methylsulfinyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide,pyridazinone derivatives, oxoprazine derivatives,N-(triazol-2-yl)arylcarboxamides, triazinones, and pyrazolones encodesthe amino acid sequence set forth in any one of SEQ ID NO:2 to NO:71,and fragments and variants thereof that encode an HPPD inhibitorherbicide tolerance polypeptide.

A. Methods for Measuring HPPD Inhibitor Tolerance

Any suitable method for measuring tolerance to HPPD inhibitor herbicidescan be used to evaluate the HPPD polypeptides of the invention.Tolerance can be measured by monitoring the ability of a cell ororganism to survive a particular HPPD inhibitor herbicide application,or the ability to carry out essential cellular functions such asphotosynthesis, protein synthesis or respiration and reproduction in amanner that is not readily discernable from untreated cells ororganisms, or the ability to have no significant difference in yield oreven improved yield for plants treated with HPPD inhibitor herbicidecompared to such plants not treated with such herbicide (but where weedshave been removed or prevented by a mechanism other than application ofthe HPPD inhibitor herbicide). In some embodiments, tolerance can bemeasured according to a visible indicator phenotype of the cell ororganism transformed with a nucleic acid comprising the gene coding forthe respective HPPD polypeptide, or in an in vitro assay of the HPPDpolypeptide, in the presence of different concentrations of the variousHPPD inhibitor herbicides. Dose responses and relative shifts in doseresponses associated with these indicator phenotypes (formation of browncolor, growth inhibition, bleaching, herbicidal effect etc.) areconveniently expressed in terms, for example, of GR50 (concentration for50% reduction of growth) or MIC (minimum inhibitory concentration)values where increases in values correspond to increases in inherenttolerance of the expressed HPPD polypeptide, in the normal manner basedupon plant damage, meristematic bleaching symptoms etc. at a range ofdifferent concentrations of herbicides. These data can be expressed interms of, for example, GR50 values derived from dose/response curveshaving “dose” plotted on the x-axis and “percentage kill”, “herbicidaleffect”, “numbers of emerging green plants” etc. plotted on the y-axiswhere increased GR50 values correspond to increased levels of inherenttolerance of the expressed HPPD polypeptide. Herbicides can suitably beapplied pre-emergence or post emergence.

In various embodiments, tolerance level of the nucleic acid or geneencoding an HPPD polypeptide according to the invention, or the HPPDpolypeptide of the invention can be screened via transgenesis,regeneration, breeding and spray testing of a test plant such astobacco, or a crop plant such as soybean, corn, or cotton. In line withthe results obtained by such screening, such plants are more tolerant,desirably tolerant to at least 2 times the normal dose recommended forfield applications, even more preferably tolerant up to 4 times thenormal dose recommended for field applications, to HPPD inhibitorherbicides (e.g. HPPD inhibitor herbicides of the class of triketones(preferably benzobicyclon, sulcotrione, mesotrione, tembotrione,tefuryltrione, bicyclopyrone, fenquinotrione), diketonitriles,isoxazoles (preferably isoxaflutole), hydroxypyrazoles (preferablypyrazoxyfen, benzofenap, pyrazolynate, pyrasulfotole, topramezone,tolpyralate), N-(1,2,5-oxadiazol-3-yl)benzamides,N-(1,3,4-oxadiazol-2-yl)benzamides (preferably2-methyl-N-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide,N-(tetrazol-5-yl)- or N-(triazol-5-yl)arylcarboxamides (preferably2-chloro-3-ethoxy-4-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide,4-(difluoromethyl)-2-methoxy-3-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide,2-chloro-3-(methylsulfanyl)-N-(1-methyl-H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide(hereinafter also named “Cmpd. 1”),2-(methoxymethyl)-3-(methylsulfinyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide,pyridazinone derivatives, oxoprazine derivatives,N-(triazol-2-yl)arylcarboxamides, triazinones, and pyrazolones than suchplants that do not contain any exogenous gene encoding an HPPDpolypeptide, or than plants that contain a gene comprising a referenceHPPD polypeptide encoding DNA, for example, a Pseudomonas fluorescensHPPD-encoding DNA, under control of the same promoter as the nucleicacid encoding the HPPD polypeptide of present invention. Accordingly,the term “capable of increasing the tolerance of a plant to at least oneherbicide acting on HPPD” denotes a tolerance by the plant expressingthe HPPD of the invention to at least 1×, 2×, or 3×, or 4×, or greater,the normal field dose of the HPPD inhibitor herbicide as compared to aplant only expressing its endogenous HPPD or a plant expressing areference HPPD polypeptide. In this regard, the term “herbicide actingon HPPD” is not limited to substances which are known and/or used asherbicides but to any substances which inhibit the catalytic activity ofHPPD polypeptides.

The term “herbicide tolerance”, “inhibitor tolerance”, or “inhibitorinsensitivity” means also the ability of an enzyme to perform itsrespective catalytic reaction in the presence of an inhibitor/herbicideor after an exposition to an inhibitor/herbicide. The herbicidetolerance of enzymes, i.e. their ability to resist the inhibitory effectof the herbicide, can be expressed qualitatively and quantitatively.Qualitatively, enzymes that tolerate different entities or evendifferent classes of inhibitors have a high tolerance and vice versa. Inquantitative terms, the tolerance of an enzyme compared to one herbicidecan be expressed as the respective “residual activity” or “residualturnover” observed in one sample of this enzyme calculated as ratio ofactivities (k_(app), kinetic measure) or total substrate turnover(change in signal, endpoint measurement) in the absence and presence ofone inhibitor (Bergmeyer, H.U.: “Methods of enzymatic analysis”, 1974).In various embodiments, for the determination of the residual activity,the apparent kinetic constant (k_(app)) of the determined substrateconversion can be measured as kinetic changes in absorbance at 320 nm ina coupled assay, in that homogentisate (HGA) formed by HPPD from HPP isdirectly converted into the well absorbing molecule maleylacetoacetate(MAA) by a second enzyme homogentisate dioxygenase (HGD), applied inexcess uniformly in all assays. The k_(cat)/k_(M) ratio of an enzymaticactivity is proportional to the apparent kinetic constant k_(app) and isproportional to k_(cat)/k_(M)*[E] ([E]=enzyme concentration). Acompetitive inhibitor exhibits an apparent increase in k_(M) and therebya reciprocal decrease in k_(app) at non-saturating substrateconcentrations. As both k_(app) measurements in the presence and absenceof inhibitor are performed by use of the identical enzyme sample, raw orpurified, and thereby at the same enzyme concentration, the enzymeconcentration eliminates from the calculation of residual activity andthe ratio of both k_(app) directly indicates the change of k_(M) due tothe inhibition. Noteworthy, this concept applies to enzyme/inhibitorpairs interacting in a “competitive inhibition” manner, probably correctfor almost all polypeptide variants and inhibitors described. Theinhibition constant ki for an enzyme and the respective inhibitordescribes the binding strength of the inhibitor to this enzyme. Anincreased tolerance is given for ratios of 1.5, 2, 3, 4, 5, 7, 10, 20,30, 40, 50, 100, 200 or higher and compared to a reference HPPD sequencein presence or absence of any respective HPPD inhibitor herbicide.

A specific, although non-limiting, type of assay that can be used toevaluate the HPPD polypeptide sequences of the invention is acolorimetric assay (as described, for example, see U.S. Pat. No.6,768,044). In this assay, for example, E. coli cells containing thevector pSE420-HPPDx (HPPDx means any gene coding for a putative HPPDpolypeptide; basic vector “pSE420” was obtained from InvitrogenKarlsruhe, Germany) or a modified version of pSE420 (pSE420(RI)NX)-HPPDxare producing soluble melanin-like pigments from the tyrosine catabolismwhen the overexpressed HPPD polypeptide is active. These melanin-likepigments are assayed in a liquid culture or by applying E. coli cultureon LB-broth type solid agar. After 16 hours to 8 days at 20-30° C., theculture medium or agar wells which have been inoculated with an E. coliculture containing the empty vector pSE420 do not alter the color of themedium, or those which have been seeded with an E. coli culturecontaining a vector pSE420-HPPDx containing a gene coding for aninactive HPPD also do not alter the color of the medium, while the wellsinoculated with an E. coli culture containing the vector pSE420-HPPDxcoding for an active HPPD are brownish. In the presence of an HPPDinhibitor herbicide, this pigment production can be inhibited and theculture will not alter the color of the medium, unless an HPPD inhibitorherbicide tolerant HPPD polypeptide is expressed and active. It has beenpreviously demonstrated that this test reflects the HPPD activity andHPPD inhibitor herbicide tolerance, whatever the origin of this activityis, and allows the identification of HPPD activities (U.S. Pat. No.6,768,044), i.e. at a qualitative level.

B. Methods of Introducing Mutations into HPPD Sequences

In the mutated HPPD polypeptides encoded by the nucleic acid of theinvention at least three amino acid have been replaced as defined above.

The replacement can be effected in the nucleic acid sequence whichencodes the reference HPPD polypeptide as defined above by any meanswhich is appropriate for replacing, in the said sequence, the codonwhich encodes the amino acid to be replaced with the codon whichcorresponds to the amino acid which is to replace it, with the saidcodons being widely described in the literature and well known to theskilled person.

Several molecular biological methods can be used to achieve thisreplacement. A useful method for preparing a mutated nucleic acidsequence according to the invention and the corresponding proteincomprises carrying out site-directed mutagenesis on codons encoding oneor more amino acids which are selected in advance. The methods forobtaining these site-directed mutations are well known to the skilledperson and widely described in the literature (in particular: DirectedMutagenesis: A Practical Approach, 1991, Edited by M. J. McPHERSON, IRLPRESS), or are methods for which it is possible to employ commercialkits (for example the QUIKCHANGE™ lightening mutagenesis kit from Qiagenor Stratagene). After the site-directed mutagenesis, it is useful toselect the cells which contain a mutated HPPD which is less sensitive toan HPPD inhibitor by using an appropriate screening aid. Appropriatescreening methods to achieve this have been described above.

Alternatively, a DNA sequence encoding the reference HPPD polypeptidecan be modified in silico to encode an HPPD polypeptide having one ormore of the substitutions recited herein, and then synthesized de novo.This method is also well known in the art, described in the literature.The nucleotide sequence encoding the mutated HPPD polypeptide can beintroduced into a host cell as described elsewhere herein.

C. Isolated Polynucleotides, and Variants and Fragments Thereof.

In some embodiments, the present invention comprises isolated orrecombinant, polynucleotides. The term “polynucleotide” corresponds toany genetic material of any length and any sequence, comprisingsingle-stranded and double-stranded DNA and RNA molecules, includingregulatory elements, structural genes, groups of genes, plasmids, wholegenomes, and fragments thereof.

A “recombinant” polynucleotide or polypeptide/protein, or biologicallyactive portion thereof, as defined herein is no longer present in itsoriginal, native organism, such as when contained in a heterologous hostcell or in a transgenic plant cell, seed or plant. In one embodiment, arecombinant polynucleotide is free of sequences (for example, proteinencoding or regulatory sequences) that naturally flank the nucleic acid(i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) inthe genomic DNA of the organism from which the polynucleotide isderived. The term “recombinant” encompasses polynucleotides orpolypeptides that have been manipulated with respect to the nativepolynucleotide or polypeptide, such that the polynucleotide orpolypeptide differs (e.g., in chemical composition or structure) fromwhat is occurring in nature. In another embodiment, a “recombinant”polynucleotide is free of internal sequences (i.e. introns) thatnaturally occur in the genomic DNA of the organism from which thepolynucleotide is derived. A typical example of such polynucleotide is aso-called Complementary DNA (cDNA). For example, in various embodiments,the isolated HPPD inhibitor herbicide tolerance-encoding polynucleotidecan contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1kb of nucleotide sequence that naturally flanks the polynucleotide ingenomic DNA of the cell from which the polynucleotide is derived.Nucleic acid molecules of the invention include those that encode theHPPD of the invention. In some embodiments, the nucleic acid molecule ofthe invention is operably linked to a promoter capable of directingexpression of the nucleic acid molecule in a host cell (e.g., a planthost cell or a bacterial host cell).

The present invention further contemplates exemplary variants andfragments of any nucleic acid sequence encoding the amino acid sequencesset forth in any of SEQ ID NO:2 to NO:71. A “fragment” of apolynucleotide may encode a biologically active portion of apolypeptide, or it may be a fragment that can be used as a hybridizationprobe or PCR primer using methods disclosed elsewhere herein.Polynucleotides that are fragments of a polynucleotide comprise at leastabout 15, 20, 50, 75, 100, 200, 300, 350, 400, 450, 500, 550, 600, 650,700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, contiguousnucleotides, or up to the number of nucleotides present in a full-lengthpolynucleotide disclosed herein depending upon the intended use (e.g.,an HPPD nucleic acid described herein). By “contiguous” nucleotides areintended nucleotide residues that are immediately adjacent to oneanother.

Fragments of the polynucleotides of the present invention generally willencode polypeptide fragments that retain the biological activity of thefull-length HPPD inhibitor herbicide tolerance protein; i.e.,herbicide-tolerance activity. By “retains herbicide tolerance activity”is intended that the fragment will have at least about 30%, at leastabout 50%, at least about 70%, at least about 80%, 85%, 90%, 95%, 100%,110%, 125%, 150%, 175%, 200%, 250%, at least about 300% or greater ofthe herbicide tolerance activity of the full-length HPPD inhibitorherbicide tolerance protein disclosed herein as SEQ ID NO:2 to NO:71.Methods for measuring herbicide tolerance activity are well known in theart and exemplary methods are described herein. In a non-limitingexample, a fragment of the invention will be tolerant to the same doseof an HPPD inhibitor herbicide, or tolerant to 1×, 2×, 3×, 4×, or higherdose of an HPPD inhibitor herbicide, or the fragments will be as or moretolerant based on ki between the fragment and SEQ ID NO:2 to NO:71.

A fragment of a polynucleotide that encodes a biologically activeportion of a polypeptide of the invention will encode at least about150, 175, 200, 250, 300, 350 contiguous amino acids, or up to the totalnumber of amino acids present in a full-length polypeptide of theinvention. In a non-limiting example, a fragment of a polynucleotidethat encodes a biologically active portion of an HPPD polypeptide having(a) an alanine at the amino acid position corresponding to amino acidposition 268 of SEQ ID NO:1, (b) a proline at the amino acid positioncorresponding to amino acid position 335 of SEQ ID NO:1, (c) a histidineor an aspartic acid at the position corresponding to amino acid position336 of SEQ ID NO:1, and (d) a serine at the position corresponding toamino acid position 337 of SEQ ID NO:1.

and, optionally, one or more further amino acid substitutions at thepositions corresponding to amino acid positions 213, 215, 270, 315, 340,344, 345 of SEQ ID NO:1, including the HPPD polypeptide set forth in anyof SEQ ID NO:2 to NO:71.

The invention also encompasses variant polynucleotides as describedsupra. “Variants” of the polynucleotide also include those sequencesthat encode the HPPD of the invention but that differ conservativelybecause of the degeneracy of the genetic code, as well as those that aresufficiently identical. Variants of the present invention will retainHPPD polypeptide activity and HPPD herbicide inhibitor tolerance. Theterm “sufficiently identical” is intended a polypeptide orpolynucleotide sequence that has at least about 53%, at least about 60%or 65% sequence identity, about 70% or 75% sequence identity, about 80%or 85% sequence identity, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% sequence identity compared to a reference sequence using oneof the alignment programs using standard parameters. One of skill in theart will recognize that these values can be appropriately adjusted todetermine corresponding identity of polypeptides encoded by twopolynucleotides by taking into account codon degeneracy, amino acidsimilarity, reading frame positioning, and the like.

Bacterial genes quite often possess multiple methionine initiationcodons in proximity to the start of the open reading frame. Often,translation initiation at one or more of these start codons will lead togeneration of a functional protein. These start codons can include ATGcodons. However, bacteria such as Bacillus sp. also recognize the codonGTG as a start codon, and proteins that initiate translation at GTGcodons contain a methionine at the first amino acid. Furthermore, it isnot often determined a priori which of these codons are used naturallyin the bacterium. Thus, it is understood that use of one of thealternate methionine codons may lead to generation of variants thatconfer herbicide tolerance. These herbicide tolerance proteins areencompassed in the present invention and may be used in the methods ofthe present invention. Naturally occurring allelic variants can beidentified with the use of well-known molecular biology techniques, suchas polymerase chain reaction (PCR) and hybridization techniques asoutlined below. Variant polynucleotides also include syntheticallyderived polynucleotides that have been generated, for example, by usingsite-directed or other mutagenesis strategies but which still encode thepolypeptide having the desired biological activity.

The skilled artisan will further appreciate that changes can beintroduced by further mutation of the polynucleotides of the inventionthereby leading to further changes in the amino acid sequence of theencoded polypeptides, without altering the biological activity of thepolypeptides. Thus, variant isolated polynucleotides can be created byintroducing one or more additional nucleotide substitutions, additions,or deletions into the corresponding polynucleotide encoding the HPPD ofthe invention, such that 3-5, 1-7, 1-9, 1-11, 1-13, 1-15, or 1-17 aminoacid substitutions, additions or deletions, or 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16 or 17 amino acid substitutions, additions ordeletions, are introduced into the encoded polypeptide. Furthermutations can be introduced by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis, or geneshuffling techniques. Such variant polynucleotides are also encompassedby the present invention.

Variant polynucleotides can be made by introducing mutations randomlyalong all or part of the coding sequence, such as by saturationmutagenesis or permutational mutagenesis, and the resultant mutants canbe screened for the ability to confer herbicide tolerance activity toidentify mutants that retain activity.

Additional methods for generating variants include subjecting a cellexpressing a protein disclosed herein (or library thereof) to a specificcondition that creates a stress to the activity of the protein. Specificconditions can include (but are not limited to) changes in temperature,changes in pH, changes in the concentrations of substrates orinhibitors, and changes in the buffer composition or theirconcentrations. The protein library can be subjected to these conditionsduring the time of protein expression (e.g. in E. coli or other host) orfollowing creation of a protein extract, or following proteinpurification.

The functional or enzymatic activity of the protein library that hasbeen subjected to a stress condition can then be compared to thereference protein to identify proteins with improved properties. Thisactivity comparison can be carried out as part of a growth screen oralternatively as part of an enzymatic assay that quantifies the activityof the protein. The properties that can be identified as improved caninclude HPPD inhibitor herbicide tolerance, changes in kinetic constants(including KM, Ki, k_(cat)), protein stability, protein thermostability,or protein temperature and pH optimum.

D. Isolated Proteins and Variants and Fragments Thereof

Herbicide tolerance polypeptides are also encompassed within the presentinvention. A herbicide tolerance polypeptide includes preparations ofpolypeptides having less than about 30%, 20%, 10%, or 5% (by dry weight)of non-herbicide tolerance polypeptide (also referred to herein as a“contaminating protein”). In the present invention, “herbicide toleranceprotein” is intended an HPPD polypeptide disclosed herein. Fragments,biologically active portions, and variants thereof are also provided,and may be used to practice the methods of the present invention.

“Fragments” or “biologically active portions” include polypeptidefragments comprising a portion of an amino acid sequence encoding anherbicide tolerance protein and that retains herbicide toleranceactivity. A biologically active portion of an herbicide toleranceprotein can be a polypeptide that is, for example, 10, 25, 50, 100 ormore amino acids in length. Such biologically active portions can beprepared by recombinant techniques and evaluated for herbicide toleranceactivity.

By “variants” is intended proteins or polypeptides having an amino acidsequence that is at least about 53%, 60%, 65%, about 70%, 75%, about80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identicalto any of the exemplary SEQ ID NO:2 to NO:71, wherein said variant hasHPPD polypeptide activity and HPPD inhibitor herbicide tolerance. One ofskill in the art will recognize that these values can be appropriatelyadjusted to determine corresponding identity of polypeptides encoded bytwo polynucleotides by taking into account codon degeneracy, amino acidsimilarity, reading frame positioning, and the like.

For example, conservative amino acid substitutions may be made at one ormore nonessential amino acid residues. A “nonessential” amino acidresidue is a residue that can be altered from the reference sequence ofa polypeptide without altering the biological activity, whereas an“essential” amino acid residue is required for biological activity. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g. lysine, arginine, histidine), acidic side chains (e.g.aspartic acid, glutamic acid), uncharged polar side chains (e.g.glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g. alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g. threonine, valine, isoleucine) and aromatic side chains(e.g. tyrosine, phenylalanine, tryptophan, histidine). Amino acidsubstitutions may be made in non-conserved regions that retain function.In general, such substitutions would not be made for conserved aminoacid residues, or for amino acid residues residing within a conservedmotif, where such residues are essential for polypeptide activity.However, one of skill in the art would understand that functionalvariants may have minor conserved or non-conserved alterations in theconserved residues.

Antibodies to the HPPD of the present invention, or to variants orfragments thereof, are also encompassed. Methods for producingantibodies are well known in the art (see, for example, Harlow and Lane(1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.; U.S. Pat. No. 4,196,265).

Thus, one aspect of the invention concerns antibodies, single-chainantigen binding molecules, or other proteins that specifically bind toone or more of the protein or peptide molecules of the invention andtheir homologs, fusions or fragments. In a particularly preferredembodiment, the antibody specifically binds to a protein having theamino acid sequence set forth in SEQ ID NO:2 to NO:71 or a fragmentthereof.

Antibodies of the invention may be used to quantitatively orqualitatively detect the protein or peptide molecules of the invention,or to detect post translational modifications of the proteins. As usedherein, an antibody or peptide is said to “specifically bind” to aprotein or peptide molecule of the invention if such binding is notcompetitively inhibited by the presence of non-related molecules.

E. Gene Stacking

In the commercial production of crops, it is desirable to eliminateunder reliable pesticidal management unwanted plants (i.e. “weeds”) froma field of crop plants. An ideal treatment would be one which could beapplied to an entire field but which would eliminate only the unwantedplants while leaving the crop plants unaffected. One such treatmentsystem would involve the use of crop plants which are tolerant to anherbicide so that when the herbicide is sprayed on a field ofherbicide-tolerant crop plants, the crop plants would continue to thrivewhile non-herbicide-tolerant weeds are killed or severely damaged.Ideally, such treatment systems would take advantage of varyingherbicide properties so that weed control could provide the bestpossible combination of flexibility and economy. For example, individualherbicides have different longevities in the field, and some herbicidespersist and are effective for a relatively long time after they areapplied to a field while other herbicides are quickly broken down intoother and/or non-active compounds. An ideal treatment system would allowthe use of different herbicides so that growers could tailor the choiceof herbicides for a particular situation.

While a number of herbicide-tolerant crop plants are presentlycommercially available, an issue that has arisen for many commercialherbicides and herbicide/crop combinations is that individual herbicidestypically have incomplete spectrum of activity against common weedspecies. For most individual herbicides which have been in use for sometime, populations of herbicide resistant weed species and biotypes havebecome more prevalent (see, e.g., Tranel and Wright (2002) Weed Science50: 700-712; Owen and Zelaya (2005) Pest Manag. Sci. 61: 301-311).Transgenic plants which are tolerant to more than one herbicide havebeen described (see, e.g. WO2005/012515). However, improvements in everyaspect of crop production, weed control options, extension of residualweed control, and improvement in crop yield are continuously in demand.

The HPPD protein or nucleotide sequence of the invention isadvantageously combined in plants with other genes which encode proteinsor RNAs that confer useful agronomic properties to such plants. Amongthe genes which encode proteins or RNAs that confer useful agronomicproperties on the transformed plants, mention can be made of the DNAsequences encoding proteins which confer tolerance to one or moreherbicides that, according to their chemical structure, differ from HPPDinhibitor herbicides, and others which confer tolerance to certaininsects, those which confer tolerance to certain diseases, DNAs thatencodes RNAs that provide nematode or insect control, and the like. Suchgenes are in particular described in published PCT Patent ApplicationsWO91/02071 and WO95/06128 and in U.S. Pat. No. 7,923,602 and US PatentApplication Publication No. 2010/0166723, each of which is hereinincorporated by reference in its entirety.

Among the DNA sequences encoding proteins which confer tolerance tocertain herbicides on the transformed plant cells and plants, mentioncan be made of a bar or PAT gene or the Streptomyces coelicolor genedescribed in WO2009/152359 which confers tolerance to glufosinateherbicides, a gene encoding a suitable EPSPS which confers tolerance toherbicides having EPSPS as a target, such as glyphosate and its salts(U.S. Pat. Nos. 4,535,060, 4,769,061, 5,094,945, 4,940,835, 5,188,642,4,971,908, 5,145,783, 5,310,667, 5,312,910, 5,627,061, 5,633,435), agene encoding glyphosate-n-acetyltransferase (for example, U.S. Pat.Nos. 8,222,489, 8,088,972, 8,044,261, 8,021,857, 8,008,547, 7,999,152,7,998,703, 7,863,503, 7,714,188, 7,709,702, 7,666,644, 7,666,643,7,531,339, 7,527,955, and 7,405,074), or a gene encoding glyphosateoxydoreductase (for example, U.S. Pat. No. 5,463,175).

Among the DNA sequences encoding a suitable EPSPS which confer toleranceto the herbicides which have EPSPS as a target, mention will moreparticularly be made of the gene which encodes a plant EPSPS, inparticular maize EPSPS, particularly a maize EPSPS which comprises twomutations, particularly a mutation at amino acid position 102 and amutation at amino acid position 106 (WO2004/074443), and which isdescribed in U.S. Pat. No. 6,566,587, hereinafter named double mutantmaize EPSPS or 2mEPSPS, or the gene which encodes an EPSPS isolated fromAgrobacterium and which is described by sequence ID No. 2 and sequenceID No. 3 of U.S. Pat. No. 5,633,435, also named CP4.

Among the DNA sequences encoding a suitable EPSPS which confer toleranceto the herbicides which have EPSPS as a target, mention will moreparticularly be made of the gene which encodes an EPSPS GRG23 fromArthrobacter globiformis, but also the mutants GRG23 ACE1, GRG23 ACE2,or GRG23 ACE3, particularly the mutants or variants of GRG23 asdescribed in WO2008/100353, such as GRG23(ace3)R173K of SEQ ID No. 29 inWO2008/100353.

In the case of the DNA sequences encoding EPSPS, and more particularlyencoding the above genes, the sequence encoding these enzymes isadvantageously preceded by a sequence encoding a transit peptide, inparticular the “optimized transit peptide” described in U.S. Pat. Nos.5,510,471 or 5,633,448.

Exemplary herbicide tolerance traits that can be combined with thenucleic acid sequence of the invention further include at least one ALS(acetolactate synthase) inhibitor (WO2007/024782); a mutated ArabidopsisALS/AHAS gene (U.S. Pat. No. 6,855,533); genes encoding2,4-D-monooxygenases conferring tolerance to 2,4-D(2,4-dichlorophenoxyacetic acid) by metabolization (U.S. Pat. No.6,153,401); and, genes encoding Dicamba monooxygenases conferringtolerance to dicamba (3,6-dichloro-2-methoxybenzoic acid) bymetabolization (US 2008/0119361 and US 2008/0120739).

In various embodiments, the HPPD of the invention is stacked with one ormore herbicide tolerant genes, including one or more additional HPPDinhibitor herbicide tolerant genes, and/or one or more genes tolerant toglyphosate and/or glufosinate. In one embodiment, the HPPD of theinvention is combined with 2mEPSPS and bar.

Among the DNA sequences encoding proteins concerning properties oftolerance to insects, mention will more particularly be made of the Btproteins widely described in the literature and well known to thoseskilled in the art. Mention will also be made of proteins extracted frombacteria such as Photorhabdus (WO97/17432 and WO98/08932).

Among such DNA sequences encoding proteins of interest which confernovel properties of tolerance to insects, mention will more particularlybe made of the Bt Cry or VIP proteins widely described in the literatureand well known to those skilled in the art. These include the Cry1Fprotein or hybrids derived from a Cry1F protein (e.g., the hybridCry1A-Cry1F proteins described in U.S. Pat. Nos. 6,326,169; 6,281,016;6,218,188, or toxic fragments thereof), the Cry1A-type proteins or toxicfragments thereof, preferably the Cry1Ac protein or hybrids derived fromthe Cry1Ac protein (e.g., the hybrid Cry1Ab-Cry1Ac protein described inU.S. Pat. No. 5,880,275) or the Cry1Ab or Bt2 protein or insecticidalfragments thereof as described in EP451878, the Cry2Ae, Cry2Af or Cry2Agproteins as described in WO2002/057664 or toxic fragments thereof, theCry1A.105 protein described in WO 2007/140256 (SEQ ID No. 7) or a toxicfragment thereof, the VIP3Aa19 protein of NCBI accession ABG20428, theVIP3Aa20 protein of NCBI accession ABG20429 (SEQ ID No. 2 in WO2007/142840), the VIP3A proteins produced in the COT202 or COT203 cottonevents (WO2005/054479 and WO2005/054480, respectively), the Cry proteinsas described in WO2001/47952, the VIP3Aa protein or a toxic fragmentthereof as described in Estruch et al. (1996), Proc Natl Acad Sci USA.28; 93(11):5389-94 and U.S. Pat. No. 6,291,156, the insecticidalproteins from Xenorhabdus (as described in WO98/50427), Serratia(particularly from S. entomophila) or Photorhabdus species strains, suchas Tc-proteins from Photorhabdus as described in WO98/08932 (e.g.,Waterfield et al., 2001, Appl Environ Microbiol. 67(11):5017-24;Ffrench-Constant and Bowen, 2000, Cell Mol Life Sci.; 57(5):828-33).Also any variants or mutants of any one of these proteins differing insome (1-10, preferably 1-5) amino acids from any of the above sequences,particularly the sequence of their toxic fragment, or which are fused toa transit peptide, such as a plastid transit peptide, or another proteinor peptide, is included herein.

In various embodiments, the HPPD sequence of the invention can becombined in plants with one or more genes conferring a desirable trait,such as herbicide tolerance, insect tolerance, drought tolerance,nematode control, water use efficiency, nitrogen use efficiency,improved nutritional value, disease resistance, improved photosynthesis,improved fiber quality, stress tolerance, improved reproduction, and thelike.

Particularly useful transgenic events which may be combined with thegenes of the current invention in plants of the same species (e.g., bycrossing or by re-transforming a plant containing another transgenicevent with a chimeric gene of the invention), include Event531/PV-GHBK04 (cotton, insect control, described in WO2002/040677),Event 1143-14A (cotton, insect control, not deposited, described inWO2006/128569); Event 1143-51B (cotton, insect control, not deposited,described in WO2006/128570); Event 1445 (cotton, herbicide tolerance,not deposited, described in US-A 2002-120964 or WO2002/034946 Event17053 (rice, herbicide tolerance, deposited as PTA-9843, described inWO2010/117737); Event 17314 (rice, herbicide tolerance, deposited asPTA-9844, described in WO2010/117735); Event 281-24-236 (cotton, insectcontrol—herbicide tolerance, deposited as PTA-6233, described inWO2005/103266 or US-A 2005-216969); Event 3006-210-23 (cotton, insectcontrol—herbicide tolerance, deposited as PTA-6233, described in US-A2007-143876 or WO2005/103266); Event 3272 (corn, quality trait,deposited as PTA-9972, described in WO2006/098952 or US-A 2006-230473);Event 33391 (wheat, herbicide tolerance, deposited as PTA-2347,described in WO2002/027004), Event 40416 (corn, insect control—herbicidetolerance, deposited as ATCC PTA-11508, described in WO 11/075593);Event 43A47 (corn, insect control—herbicide tolerance, deposited as ATCCPTA-11509, described in WO2011/075595); Event 5307 (corn, insectcontrol, deposited as ATCC PTA-9561, described in WO2010/077816); EventASR-368 (bent grass, herbicide tolerance, deposited as ATCC PTA-4816,described in US-A 2006-162007 or WO2004/053062); Event B16 (corn,herbicide tolerance, not deposited, described in US-A 2003-126634);Event BPS-CV127-9 (soybean, herbicide tolerance, deposited as NCIMB No.41603, described in WO2010/080829); Event BLR1 (oilseed rape,restoration of male sterility, deposited as NCIMB 41193, described inWO2005/074671); Event CE43-67B (cotton, insect control, deposited as DSMACC2724, described in US-A 2009-217423 or WO2006/128573); Event CE44-69D(cotton, insect control, not deposited, described in US-A 2010-0024077);Event CE44-69D (cotton, insect control, not deposited, described inWO2006/128571); Event CE46-02A (cotton, insect control, not deposited,described in WO2006/128572); Event COT102 (cotton, insect control, notdeposited, described in US-A 2006-130175 or WO2004/039986); Event COT202(cotton, insect control, not deposited, described in US-A 2007-067868 orWO2005/054479); Event COT203 (cotton, insect control, not deposited,described in WO2005/054480); Event DAS21606-3/1606 (soybean, herbicidetolerance, deposited as PTA-11028, described in WO2012/033794), EventDAS40278 (corn, herbicide tolerance, deposited as ATCC PTA-10244,described in WO2011/022469); Event DAS-44406-6/pDAB8264.44.06.1(soybean, herbicide tolerance, deposited as PTA-11336, described inWO2012/075426), Event DAS-14536-7/pDAB8291.45.36.2 (soybean, herbicidetolerance, deposited as PTA-11335, described in WO2012/075429), EventDAS-59122-7 (corn, insect control—herbicide tolerance, deposited as ATCCPTA 11384, described in US-A 2006-070139); Event DAS-59132 (corn, insectcontrol—herbicide tolerance, not deposited, described in WO2009/100188);Event DAS68416 (soybean, herbicide tolerance, deposited as ATCCPTA-10442, described in WO2011/066384 or WO2011/066360); EventDP-098140-6 (corn, herbicide tolerance, deposited as ATCC PTA-8296,described in US-A 2009-137395 or WO 08/112019); Event DP-305423-1(soybean, quality trait, not deposited, described in US-A 2008-312082 orWO2008/054747); Event DP-32138-1 (corn, hybridization system, depositedas ATCC PTA-9158, described in US-A 2009-0210970 or WO2009/103049);Event DP-356043-5 (soybean, herbicide tolerance, deposited as ATCCPTA-8287, described in US-A 2010-0184079 or WO2008/002872); Event EE-1(brinjal, insect control, not deposited, described in WO 07/091277);Event FI117 (corn, herbicide tolerance, deposited as ATCC 209031,described in US-A 2006-059581 or WO 98/044140); Event FG72 (soybean,herbicide tolerance, deposited as PTA-11041, described inWO2011/063413), Event GA21 (corn, herbicide tolerance, deposited as ATCC209033, described in US-A 2005-086719 or WO 98/044140); Event GG25(corn, herbicide tolerance, deposited as ATCC 209032, described in US-A2005-188434 or WO 98/044140); Event GHB119 (cotton, insectcontrol-herbicide tolerance, deposited as ATCC PTA-8398, described inWO2008/151780); Event GHB614 (cotton, herbicide tolerance, deposited asATCC PTA-6878, described in US-A 2010-050282 or WO2007/017186); EventGJ11 (corn, herbicide tolerance, deposited as ATCC 209030, described inUS-A 2005-188434 or WO98/044140); Event GM RZ13 (sugar beet, virusresistance, deposited as NCIMB-41601, described in WO2010/076212); EventH7-1 (sugar beet, herbicide tolerance, deposited as NCIMB 41158 or NCIMB41159, described in US-A 2004-172669 or WO 2004/074492); Event JOPLINI(wheat, disease tolerance, not deposited, described in US-A2008-064032); Event LL27 (soybean, herbicide tolerance, deposited asNCIMB41658, described in WO2006/108674 or US-A 2008-320616); Event LL55(soybean, herbicide tolerance, deposited as NCIMB 41660, described in WO2006/108675 or US-A 2008-196127); Event LLcotton25 (cotton, herbicidetolerance, deposited as ATCC PTA-3343, described in WO2003/013224 orUS-A 2003-097687); Event LLRICE06 (rice, herbicide tolerance, depositedas ATCC 203353, described in U.S. Pat. No. 6,468,747 or WO2000/026345);Event LLRice62 (rice, herbicide tolerance, deposited as ATCC 203352,described in WO2000/026345), Event LLRICE601 (rice, herbicide tolerance,deposited as ATCC PTA-2600, described in US-A 2008-2289060 orWO2000/026356); Event LY038 (corn, quality trait, deposited as ATCCPTA-5623, described in US-A 2007-028322 or WO2005/061720); Event MIR162(corn, insect control, deposited as PTA-8166, described in US-A2009-300784 or WO2007/142840); Event MIR604 (corn, insect control, notdeposited, described in US-A 2008-167456 or WO2005/103301); EventMON15985 (cotton, insect control, deposited as ATCC PTA-2516, describedin US-A 2004-250317 or WO2002/100163); Event MON810 (corn, insectcontrol, not deposited, described in US-A 2002-102582); Event MON863(corn, insect control, deposited as ATCC PTA-2605, described inWO2004/011601 or US-A 2006-095986); Event MON87427 (corn, pollinationcontrol, deposited as ATCC PTA-7899, described in WO2011/062904); EventMON87460 (corn, stress tolerance, deposited as ATCC PTA-8910, describedin WO2009/111263 or US-A 2011-0138504); Event MON87701 (soybean, insectcontrol, deposited as ATCC PTA-8194, described in US-A 2009-130071 orWO2009/064652); Event MON87705 (soybean, quality trait—herbicidetolerance, deposited as ATCC PTA-9241, described in US-A 2010-0080887 orWO2010/037016); Event MON87708 (soybean, herbicide tolerance, depositedas ATCC PTA-9670, described in WO2011/034704); Event MON87712 (soybean,yield, deposited as PTA-10296, described in WO2012/051199), EventMON87754 (soybean, quality trait, deposited as ATCC PTA-9385, describedin WO2010/024976); Event MON87769 (soybean, quality trait, deposited asATCC PTA-8911, described in US-A 2011-0067141 or WO2009/102873); EventMON88017 (corn, insect control-herbicide tolerance, deposited as ATCCPTA-5582, described in US-A 2008-028482 or WO2005/059103); EventMON88913 (cotton, herbicide tolerance, deposited as ATCC PTA-4854,described in WO2004/072235 or US-A 2006-059590); Event MON88302 (oilseedrape, herbicide tolerance, deposited as PTA-10955, described inWO2011/153186), Event MON88701 (cotton, herbicide tolerance, depositedas PTA-11754, described in WO2012/134808), Event MON89034 (corn, insectcontrol, deposited as ATCC PTA-7455, described in WO 07/140256 or US-A2008-260932); Event MON89788 (soybean, herbicide tolerance, deposited asATCC PTA-6708, described in US-A 2006-282915 or WO2006/130436); EventMS11 (oilseed rape, pollination control—herbicide tolerance, depositedas ATCC PTA-850 or PTA-2485, described in WO2001/031042); Event MS8(oilseed rape, pollination control—herbicide tolerance, deposited asATCC PTA-730, described in WO2001/041558 or US-A 2003-188347); EventNK603 (corn, herbicide tolerance, deposited as ATCC PTA-2478, describedin US-A 2007-292854); Event PE-7 (rice, insect control, not deposited,described in WO2008/114282); Event RF3 (oilseed rape, pollinationcontrol—herbicide tolerance, deposited as ATCC PTA-730, described inWO2001/041558 or US-A 2003-188347); Event RT73 (oilseed rape, herbicidetolerance, not deposited, described in WO2002/036831 or US-A2008-070260); Event SYHTOH2/SYN-000H2-5 (soybean, herbicide tolerance,deposited as PTA-11226, described in WO2012/082548), Event T227-1 (sugarbeet, herbicide tolerance, not deposited, described in WO2002/44407 orUS-A 2009-265817); Event T25 (corn, herbicide tolerance, not deposited,described in US-A 2001-029014 or WO2001/051654); Event T304-40 (cotton,insect control—herbicide tolerance, deposited as ATCC PTA-8171,described in US-A 2010-077501 or WO2008/122406); Event T342-142 (cotton,insect control, not deposited, described in WO2006/128568); Event TC1507(corn, insect control—herbicide tolerance, not deposited, described inUS-A 2005-039226 or WO2004/099447); Event VIP1034 (corn, insectcontrol—herbicide tolerance, deposited as ATCC PTA-3925., described inWO2003/052073), Event 32316 (corn, insect control-herbicide tolerance,deposited as PTA-11507, described in WO2011/084632), Event 4114 (corn,insect control-herbicide tolerance, deposited as PTA-11506, described inWO2011/084621), event EE-GM3/FG72 (soybean, herbicide tolerance, ATCCAccession N^(o) PTA-11041) optionally stacked with event EE-GM1/LL27 orevent EE-GM2/LL55 (WO2011/063413A2), event DAS-68416-4 (soybean,herbicide tolerance, ATCC Accession N^(o) PTA-10442, WO2011/066360A1),event DAS-68416-4 (soybean, herbicide tolerance, ATCC Accession N^(o)PTA-10442, WO2011/066384A1), event DP-040416-8 (corn, insect control,ATCC Accession N^(o) PTA-11508, WO2011/075593A1), event DP-043A47-3(corn, insect control, ATCC Accession N^(o) PTA-11509, WO2011/075595A1),event DP-004114-3 (corn, insect control, ATCC Accession N^(o) PTA-11506,WO2011/084621A1), event DP-032316-8 (corn, insect control, ATCCAccession N^(o) PTA-11507, WO2011/084632A1), event MON-88302-9 (oilseedrape, herbicide tolerance, ATCC Accession N^(o) PTA-10955,WO2011/153186A1), event DAS-21606-3 (soybean, herbicide tolerance, ATCCAccession No. PTA-1 1028, WO2012/033794A2), event MON-87712-4 (soybean,quality trait, ATCC Accession N^(o). PTA-10296, WO2012/051199A2), eventDAS-44406-6 (soybean, stacked herbicide tolerance, ATCC Accession N^(o).PTA-11336, WO2012/075426A1), event DAS-14536-7 (soybean, stackedherbicide tolerance, ATCC Accession N^(o). PTA-11335, WO2012/075429A1),event SYN-000H2-5 (soybean, herbicide tolerance, ATCC Accession N^(o).PTA-11226, WO2012/082548A2), event DP-061061-7 (oilseed rape, herbicidetolerance, no deposit N^(o) available, WO2012071039A1), eventDP-073496-4 (oilseed rape, herbicide tolerance, no deposit N^(o)available, US2012131692), event 8264.44.06.1 (soybean, stacked herbicidetolerance, Accession N^(o) PTA-11336, WO2012/075426A2), event8291.45.36.2 (soybean, stacked herbicide tolerance, Accession N^(o).PTA-11335, WO2012/075429A2), event SYHTOH2 (soybean, ATCC AccessionN^(o). PTA-11226, WO2012/082548A2), event MON88701 (cotton, ATCCAccession N^(o) PTA-11754, WO2012/134808A1), event KK179-2 (alfalfa,ATCC Accession N^(o) PTA-11833, WO2013/003558A1), event pDAB8264.42.32.1(soybean, stacked herbicide tolerance, ATCC Accession N^(o) PTA-11993,WO2013/010094A1), event MZDT09Y (corn, ATCC Accession N^(o) PTA-13025,WO2013/012775A1), event 4114 (Maize, insect control, ATCC AccessionN^(o) PTA-11506) WO2013/14901, event MON87411 (Maize, ATCC AccessionN^(o) PTA-12669) WO2013/169923, event A26-5 (Cotton, insect control)WO2013/170398, event A2-6 (Cotton, insect control) WO2013/170399, event9582.816.15.1 (Soybean, insect control, herbicide tolerance), ATCCAccession N^(o) PTA-12588) WO2014/004458, event 33121 (Maize, insectcontrol, herbicide tolerance, ATCC Accession N^(o) PTA-13392)WO2014/116854, event 32218 (Maize insect control, herbicide tolerance,ATCC Accession N^(o) PTA-13391) WO2014/116989, event “SPT-7R-949DSPT-7R-1425D” (Rice male sterility) WO2014/154115, event MON87751(Soybean, ATCC Accession N^(o). PYA-120166) WO2014/201235, event“Pp009-401 Pp009-415 Pp009-469” (Turfgrass, ATCC Accession N^(o)PTA-120354, PTA-120353, PTA-120355) WO2015006774, event Bs2-X5 (Tomato,ATCC) WO2015/017637, event MON87403 (Maize, grain yield, ATCC AccessionN^(o) PTA-13584 WO2015/053998, event 32218 (Maize, insect control, ATCCAccession N^(o) PTA-13391) WO2015/112182.

F. Polynucleotide Constructs

The polynucleotides encoding the HPPD polypeptides of the presentinvention may be modified to obtain or enhance expression in plantcells. The polynucleotides encoding the polypeptides identified hereinmay be provided in expression cassettes for expression in the plant ofinterest. A “plant expression cassette” includes a DNA construct,including a recombinant DNA construct, that is capable of resulting inthe expression of a polynucleotide in a plant cell. The cassette caninclude in the 5′-3′ direction of transcription, a transcriptionalinitiation region (i.e. promoter, particularly a heterologous promoter)operably-linked to one or more polynucleotides of interest, and/or atranslation and transcriptional termination region (i.e. terminationregion) functional in plants. The cassette may additionally contain atleast one additional polynucleotide to be introduced into the organism,such as a selectable marker gene. Alternatively, the additionalpolynucleotide(s) can be provided on multiple expression cassettes. Suchan expression cassette is provided with a plurality of restriction sitesfor insertion of the polynucleotide(s) to be under the transcriptionalregulation of the regulatory regions.

In a further embodiment, the present invention relates to a chimericgene comprising a coding sequence comprising heterologous the nucleicacid of the invention operably linked to a plant-expressible promoterand optionally a transcription termination and polyadenylation region.“Heterologous” generally refers to the polynucleotide or polypeptidethat is not endogenous to the cell or is not endogenous to the locationin the native genome in which it is present, and has been added to thecell by infection, transfection, microinjection, electroporation,microprojection, or the like. By “operably linked” is intended afunctional linkage between two polynucleotides. For example, when apromoter is operably linked to a DNA sequence, the promoter sequenceinitiates and mediates transcription of the DNA sequence. It isrecognized that operably linked polynucleotides may or may not becontiguous and, where used to reference the joining of two polypeptidecoding regions, the polypeptides are expressed in the same readingframe.

The promoter may be any polynucleotide sequence which showstranscriptional activity in the chosen plant cells, plant parts, orplants. The promoter may be native or analogous, or foreign orheterologous, to the plant host and/or to the DNA sequence of theinvention. Where the promoter is “native” or “analogous” to the planthost, it is intended that the promoter is found in the native plant intowhich the promoter is introduced. Where the promoter is “foreign” or“heterologous” to the DNA sequence of the invention, it is intended thatthe promoter is not the native or naturally occurring promoter for theoperably linked DNA sequence of the invention. The promoter may beinducible or constitutive. It may be naturally-occurring, may becomposed of portions of various naturally-occurring promoters, or may bepartially or totally synthetic. Guidance for the design of promoters isprovided by studies of promoter structure, such as that of Harley andReynolds (1987) Nucleic Acids Res. 15:2343-2361. Also, the location ofthe promoter relative to the transcription start may be optimized. See,e.g., Roberts et al. (1979) Proc. Natl. Acad. Sci. USA, 76:760-764. Manysuitable promoters for use in plants are well known in the art.

For instance, suitable constitutive promoters for use in plants include:the promoters from plant viruses, such as the peanut chlorotic streakcaulimovirus (PC1SV) promoter (U.S. Pat. No. 5,850,019); the 35Spromoter from cauliflower mosaic virus (CaMV) (Odell et al. (1985)Nature 313:810-812); the 35S promoter described in Kay et al. (1987)Science 236: 1299-1302; promoters of Chlorella virus methyltransferasegenes (U.S. Pat. No. 5,563,328) and the full-length transcript promoterfrom figwort mosaic virus (FMV) (U.S. Pat. No. 5,378,619); the promotersfrom genes such as rice actin (McElroy et al. (1990) Plant Cell2:163-171 and U.S. Pat. No. 5,641,876); ubiquitin (Christensen et al.(1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) PlantMol. Biol. 18:675-689) and Grefen et al. (2010) Plant J, 64:355-365;pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten etal. (1984) EMBO J. 3:2723-2730 and U.S. Pat. No. 5,510,474); maize H3histone (Lepetit et al. (1992) Mol. Gen. Genet. 231:276-285 andAtanassova et al. (1992) Plant J. 2(3):291-300); Brassica napus ALS3(PCT application WO97/41228); a plant ribulose-biscarboxylase/oxygenase(RuBisCO) small subunit gene; the circovirus (AU 689 311) or the Cassavavein mosaic virus (CsVMV, U.S. Pat. No. 7,053,205); and promoters ofvarious Agrobacterium genes (see U.S. Pat. Nos. 4,771,002; 5,102,796;5,182,200; and 5,428,147).

Suitable inducible promoters for use in plants include: the promoterfrom the ACE1 system which responds to copper (Mett et al. (1993) PNAS90:4567-4571); the promoter of the maize In2 gene which responds tobenzenesulfonamide herbicide safeners (Hershey et al. (1991) Mol. Gen.Genetics 227:229-237 and Gatz et al. (1994) Mol. Gen. Genetics243:32-38); and the promoter of the Tet repressor from Tn10 (Gatz et al.(1991) Mol. Gen. Genet. 227:229-237). Another inducible promoter for usein plants is one that responds to an inducing agent to which plants donot normally respond. An exemplary inducible promoter of this type isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone (Schena etal. (1991) Proc. Natl. Acad. Sci. USA 88:10421) or the recentapplication of a chimeric transcription activator, XVE, for use in anestrogen receptor-based inducible plant expression system activated byestradiol (Zuo et al. (2000) Plant J., 24:265-273). Other induciblepromoters for use in plants are described in EP 332104, PCT WO 93/21334and PCT WO 97/06269 which are herein incorporated by reference in theirentirety. Promoters composed of portions of other promoters andpartially or totally synthetic promoters can also be used. See, e.g., Niet al. (1995) Plant J. 7:661-676 and PCT WO 95/14098 describing suchpromoters for use in plants.

In one embodiment of this invention, a promoter sequence specific forparticular regions or tissues of plants can be used to express the HPPDproteins of the invention, such as promoters specific for seeds (Datla,R. et al., 1997, Biotechnology Ann. Rev. 3, 269-296), especially thenapin promoter (EP 255 378 A1), the phaseolin promoter, the gluteninpromoter, the helianthinin promoter (WO92/17580), the albumin promoter(WO98/45460), the oleosin promoter (WO98/45461), the SAT1 promoter orthe SAT3 promoter (PCT/US98/06978).

Use may also be made of an inducible promoter advantageously chosen fromthe phenylalanine ammonia lyase (PAL), HMG-CoA reductase (HMG),chitinase, glucanase, proteinase inhibitor (PI), PR1 family gene,nopaline synthase (nos) and vspB promoters (U.S. Pat. No. 5,670,349,Table 3), the HMG2 promoter (U.S. Pat. No. 5,670,349), the applebeta-galactosidase (ABG1) promoter and the apple aminocyclopropanecarboxylate synthase (ACC synthase) promoter (WO98/45445). Multiplepromoters can be used in the constructs of the invention, including insuccession.

The promoter may include, or be modified to include, one or moreenhancer elements. In some embodiments, the promoter may include aplurality of enhancer elements. Promoters containing enhancer elementsprovide for higher levels of transcription as compared to promoters thatdo not include them. Suitable enhancer elements for use in plantsinclude the PC1SV enhancer element (U.S. Pat. No. 5,850,019), the CaMV35S enhancer element (U.S. Pat. Nos. 5,106,739 and 5,164,316) and theFMV enhancer element (Maiti et al. (1997) Transgenic Res. 6:143-156);the translation activator of the tobacco mosaic virus (TMV) described inApplication WO87/07644, or of the tobacco etch virus (TEV) described byCarrington & Freed 1990, J. Virol. 64: 1590-1597, for example, orintrons such as the adhl intron of maize or intron 1 of rice actin. Seealso PCT WO96/23898, WO2012/021794, WO2012/021797, WO2011/084370, andWO2011/028914.

Often, such constructs can contain 5′ and 3′ untranslated regions. Suchconstructs may contain a “signal sequence” or “leader sequence” tofacilitate co-translational or post-translational transport of thepeptide of interest to certain intracellular structures such as thechloroplast (or other plastid), endoplasmic reticulum, or Golgiapparatus, or to be secreted. For example, the construct can beengineered to contain a signal peptide to facilitate transfer of thepeptide to the endoplasmic reticulum. By “signal sequence” is intended asequence that is known or suspected to result in co-translational orpost-translational peptide transport across the cell membrane. Ineukaryotes, this typically involves secretion into the Golgi apparatus,with some resulting glycosylation. By “leader sequence” is intended anysequence that, when translated, results in an amino acid sequencesufficient to trigger co-translational transport of the peptide chain toa sub-cellular organelle. Thus, this includes leader sequences targetingtransport and/or glycosylation by passage into the endoplasmicreticulum, passage to vacuoles, plastids including chloroplasts,mitochondria, and the like. It may also be preferable to engineer theplant expression cassette to contain an intron, such that mRNAprocessing of the intron is required for expression.

By “3′ untranslated region” is intended a polynucleotide locateddownstream of a coding sequence. Polyadenylation signal sequences andother sequences encoding regulatory signals capable of affecting theaddition of polyadenylic acid tracts to the 3′ end of the mRNA precursorare 3′ untranslated regions. By “5′ untranslated region” is intended apolynucleotide located upstream of a coding sequence.

Other upstream or downstream untranslated elements include enhancers.Enhancers are polynucleotides that act to increase the expression of apromoter region. Enhancers are well known in the art and include, butare not limited to, the SV40 enhancer region and the 35S enhancerelement.

The termination region may be native with the transcriptional initiationregion, may be native with the sequence of the present invention, or maybe derived from another source. Convenient termination regions areavailable from the Ti-plasmid of A. tumefaciens, such as the octopinesynthase and nopaline synthase termination regions. See also Guerineauet al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al.(1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158;Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; Joshi et al.(1987) Nucleic Acid Res. 15:9627-9639; and European Patent ApplicationEP 0 633 317 A1.

In one aspect of the invention, synthetic DNA sequences are designed fora given polypeptide, such as the polypeptides of the invention.Expression of the open reading frame of the synthetic DNA sequence in acell results in production of the polypeptide of the invention.Synthetic DNA sequences can be useful to simply remove unwantedrestriction endonuclease sites, to facilitate DNA cloning strategies, toalter or remove any potential codon bias, to alter or improve GCcontent, to remove or alter alternate reading frames, and/or to alter orremove intron/exon splice recognition sites, polyadenylation sites,Shine-Delgarno sequences, unwanted promoter elements and the like thatmay be present in a native DNA sequence. It is also possible thatsynthetic DNA sequences may be utilized to introduce other improvementsto a DNA sequence, such as introduction of an intron sequence, creationof a DNA sequence that is expressed as a protein fusion to organelletargeting sequences, such as chloroplast transit peptides,apoplast/vacuolar targeting peptides, or peptide sequences that resultin retention of the resulting peptide in the endoplasmic reticulum.Synthetic genes can also be synthesized using host cell-preferred codonsfor improved expression, or may be synthesized using codons at ahost-preferred codon usage frequency. See, for example, Campbell andGowri (1990) Plant Physiol. 92:1-11; U.S. Pat. Nos. 6,320,100;6,075,185; 5,380,831; and 5,436,391, U.S. Published Application Nos.20040005600 and 20010003849, and Murray et al. (1989) Nucleic Acids Res.17:477-498, herein incorporated by reference.

In one embodiment, the polynucleotides of interest are targeted to thechloroplast for expression. In this manner, where the polynucleotide ofinterest is not directly inserted into the chloroplast, the expressioncassette will additionally contain a polynucleotide encoding a transitpeptide to direct the nucleotide of interest to the chloroplasts. Suchtransit peptides are known in the art. See, for example, Von Heijne etal. (1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol.Chem. 264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol.84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Conmmun.196:1414-1421; and Shah et al. (1986) Science 233:478-481.

The polynucleotides of interest to be targeted to the chloroplast may beoptimized for expression in the chloroplast to account for differencesin codon usage between the plant nucleus and this organelle. In thismanner, the polynucleotides of interest may be synthesized usingchloroplast-preferred codons. See, for example, U.S. Pat. No. 5,380,831,herein incorporated by reference.

This plant expression cassette can be inserted into a planttransformation vector. By “transformation vector” is intended a DNAmolecule that allows for the transformation of a cell. Such a moleculemay consist of one or more expression cassettes, and may be organizedinto more than one vector DNA molecule. For example, binary vectors areplant transformation vectors that utilize two non-contiguous DNA vectorsto encode all requisite cis- and trans-acting functions fortransformation of plant cells (Hellens and Mullineaux (2000) Trends inPlant Science 5:446-451). “Vector” refers to a polynucleotide constructdesigned for transfer between different host cells. “Expression vector”refers to a vector that has the ability to incorporate, integrate andexpress heterologous DNA sequences or fragments in a foreign cell.

The plant transformation vector comprises one or more DNA vectors forachieving plant transformation. For example, it is a common practice inthe art to utilize plant transformation vectors that comprise more thanone contiguous DNA segment. These vectors are often referred to in theart as binary vectors. Binary vectors as well as vectors with helperplasmids are most often used for Agrobacterium-mediated transformation,where the size and complexity of DNA segments needed to achieveefficient transformation is quite large, and it is advantageous toseparate functions onto separate DNA molecules. Binary vectors typicallycontain a plasmid vector that contains the cis-acting sequences requiredfor T-DNA transfer (such as left border and right border), a selectablemarker that is engineered to be capable of expression in a plant cell,and a “polynucleotide of interest” (a polynucleotide engineered to becapable of expression in a plant cell for which generation of transgenicplants is desired). Also present on this plasmid vector are sequencesrequired for bacterial replication. The cis-acting sequences arearranged in a fashion to allow efficient transfer into plant cells andexpression therein. For example, the selectable marker sequence and thesequence of interest are located between the left and right borders.Often a second plasmid vector contains the trans-acting factors thatmediate T-DNA transfer from Agrobacterium to plant cells. This plasmidoften contains the virulence functions (Vir genes) that allow infectionof plant cells by Agrobacterium, and transfer of DNA by cleavage atborder sequences and vir-mediated DNA transfer, as is understood in theart (Hellens and Mullineaux (2000) Trends in Plant Science, 5:446-451).Several types of Agrobacterium strains (e.g., LBA4404, GV3101, EHA101,EHA105, etc.) can be used for plant transformation. The second plasmidvector is not necessary for introduction of polynucleotides into plantsby other methods such as microprojection, microinjection,electroporation, polyethylene glycol, etc.

G. Plant Transformation

Methods of the invention involve introducing a nucleotide construct intoa plant. By “introducing” is intended to present to the plant thenucleotide construct in such a manner that the construct gains access tothe interior of a cell of the plant. The methods of the invention do notrequire that a particular method for introducing a nucleotide constructto a plant is used, only that the nucleotide construct gains access tothe interior of at least one cell of the plant. Methods for introducingnucleotide constructs into plants are known in the art including, butnot limited to, stable transformation methods, transient transformationmethods, and virus-mediated methods. See, for example, the methods fortransforming plant cells and regenerating plants described in: U.S. Pat.Nos. 4,459,355, 4,536,475, 5,464,763, 5,177,010, 5,187,073, EP 267,159A1, EP 604 662 A1, EP 672 752 A1, U.S. Pat. Nos. 4,945,050, 5,036,006,5,100,792, 5,371,014, 5,478,744, 5,179,022, 5,565,346, 5,484,956,5,508,468, 5,538,877, 5,554,798, 5,489,520, 5,510,318, 5,204,253,5,405,765, EP 442 174 A1, EP 486 233 A1, EP 486 234 A1, EP 539 563 A1,EP 674 725 A1, WO91/02071, WO95/06128, WO2011/095460, WO2012006439A2,WO2012/006443A2, WO2012/015039A1, WO2012/019660A1, WO2012/021494A1,WO2012/064827A1, WO2012/075562A1, WO2012/077664A1, WO2012/083137A1,WO2012/084962A1, WO2012/092577A1, WO2012/109947A1, WO2012/129443A2,WO2012/138629A2, WO2012/139416A1, WO2012/149011A1, WO2013/014585A1,WO2013/025670A1, WO2013/033308A2, WO2013/066007A1, WO2013/077420A1,WO2013/090734A1, WO2013/149726A1, WO2013/180311A1, WO2014/029044A1,WO2014/029045A1, WO2014/062036A1, WO2014/065857A1, WO2014/100234A1,WO2014/100406A1, WO2014/123208A1, WO2014/143304A1, WO2014/144513A2,WO2014/157541A1, WO2014/200842A2, WO2015/051083A1, WO2015/077620A1,WO2015/085990A1, WO2015/099674A1, WO2015/118640A1, WO2015/119166A1, eachof which is herein incorporated by reference, particularly with respectto the transformation methods described therein.

In general, plant transformation methods involve transferringheterologous DNA into target plant cells (e.g. immature or matureembryos, suspension cultures, undifferentiated callus, protoplasts,etc.), followed by applying a maximum threshold level of appropriateselection (depending on the selectable marker gene) to recover thetransformed plant cells from a group of untransformed cell mass.Explants are typically transferred to a fresh supply of the same mediumand cultured routinely. Subsequently, the transformed cells aredifferentiated into shoots after placing on regeneration mediumsupplemented with a maximum threshold level of selecting agent. Theshoots are then transferred to a selective rooting medium for recoveringrooted shoot or plantlet. The transgenic plantlet then grow into matureplants and produce fertile seeds (e.g. Hiei et al. (1994) The PlantJournal 6:271-282; Ishida et al. (1996) Nature Biotechnology14:745-750). Explants are typically transferred to a fresh supply of thesame medium and cultured routinely. A general description of thetechniques and methods for generating transgenic plants are found inAyres and Park (1994) Critical Reviews in Plant Science 13:219-239 andBommineni and Jauhar (1997) Maydica 42:107-120. Since the transformedmaterial contains many cells; both transformed and non-transformed cellsare present in any piece of subjected target callus or tissue or groupof cells. The ability to kill non-transformed cells and allowtransformed cells to proliferate results in transformed plant cultures.Often, the ability to remove non-transformed cells is a limitation torapid recovery of transformed plant cells and successful generation oftransgenic plants. Molecular and biochemical methods can be used toconfirm the presence of the integrated heterologous gene of interest inthe genome of transgenic plant.

Generation of transgenic plants may be performed by one of severalmethods, including, but not limited to, introduction of heterologous DNAby Agrobacterium into plant cells (Agrobacterium-mediatedtransformation), bombardment of plant cells with heterologous foreignDNA adhered to particles, and various other non-particle direct-mediatedmethods (e.g. Hiei et al. (1994) The Plant Journal 6:271-282; Ishida etal. (1996) Nature Biotechnology 14:745-750; Ayres and Park (1994)Critical Reviews in Plant Science 13:219-239; Bommineni and Jauhar(1997) Maydica 42:107-120) to transfer DNA.

Methods for transformation of chloroplasts are known in the art. See,for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530;Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab andMaliga (1993) EMBO J. 12:601-606. The method relies on particle gundelivery of DNA containing a selectable marker and targeting of the DNAto the plastid genome through homologous recombination. Additionally,plastid transformation can be accomplished by transactivation of asilent plastid-borne transgene by tissue-preferred expression of anuclear-encoded and plastid-directed RNA polymerase. Such a system hasbeen reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA91:7301-7305.

The plant cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present invention provides transformed seed (alsoreferred to as “transgenic seed”) having a nucleotide construct of theinvention, for example, an expression cassette of the invention, stablyincorporated into their genome. In various embodiments, the seed can becoated with at least one fungicide and/or at least one insecticide, atleast one herbicide, and/or at least one safener, or any combinationthereof.

H Evaluation of Plant Transformation

Following introduction of heterologous foreign DNA into plant cells, thetransformation or integration of the heterologous gene in the plantgenome is confirmed by various methods such as analysis of nucleicacids, proteins and metabolites associated with the integrated gene.

PCR analysis is a rapid method to screen transformed cells, tissue orshoots for the presence of incorporated gene at the earlier stage beforetransplanting into the soil (Sambrook and Russell (2001) MolecularCloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.)). PCR is carried out using oligonucleotide primersspecific to the gene of interest or Agrobacterium vector background,etc.

Plant transformation may be confirmed by Southern blot analysis ofgenomic DNA (Sambrook and Russell (2001) supra). In general, total DNAis extracted from the transformant, digested with appropriaterestriction enzymes, fractionated in an agarose gel and transferred to anitrocellulose or nylon membrane. The membrane or “blot” can then beprobed with, for example, radiolabeled ³²P target DNA fragment toconfirm the integration of the introduced gene in the plant genomeaccording to standard techniques (Sambrook and Russell, 2001, supra).

In Northern analysis, RNA is isolated from specific tissues oftransformant, fractionated in a formaldehyde agarose gel, and blottedonto a nylon filter according to standard procedures that are routinelyused in the art (Sambrook and Russell (2001) supra). Expression of RNAencoded by nucleotide sequences of the invention is then tested byhybridizing the filter to a radioactive probe derived by methods knownin the art (Sambrook and Russell (2001) supra). RNA can also be detectedand/or quantified using reverse transcriptase PCR as known in the art(e.g., Green and Sambrook (2012) Molecular Cloning: A Laboratory Manual,4^(th) Edition, Cold Spring Harbor Laboratory Press, Woodbury, N.Y.).

Western blot, ELISA, lateral flow testing, and biochemical assays andthe like may be carried out on the transgenic plants to determine thepresence of protein encoded by the herbicide tolerance gene by standardprocedures (Sambrook and Russell (2001) supra) using antibodies thatbind to one or more epitopes present on the herbicide tolerance protein.

In one aspect of the invention, the HPPD genes described herein areuseful as markers to assess transformation of bacterial or plant cells.

I. Use as a Marker for Transformation

The invention also relates to the use, in a method for transformingplants, of a nucleic acid which encodes an HPPD according to theinvention as a marker gene or as a coding sequence which makes itpossible to confer to the plant tolerance to herbicides which are HPPDinhibitors, and the use of one or more HPPD inhibitor(s) on plantscomprising a nucleic acid sequence encoding an HPPD according to theinvention. See, for example, U.S. Pat. No. 6,791,014, which is hereinincorporated by reference in its entirety.

In this embodiment, an HPPD inhibitor can be introduced into the culturemedium of the competent plant cells so as to bleach said cells beforethe transformation step. The bleached competent cells are thentransformed with the gene for tolerance to HPPD inhibitors, as aselection marker, and the transformed cells which have integrated saidselection marker into their genome become green, enabling them to beselected. Such a process makes it possible to decrease the time requiredfor selecting the transformed cells.

Thus, one embodiment of the present invention consists of a method fortransforming plant cells by introducing a heterologous gene into saidplant cells with a gene for tolerance to HPPD inhibitors as selectionmarkers, wherein the method comprises preparing and culturing competentplant cells capable of receiving the heterologous gene in a suitablemedium and introducing a suitable amount of HPPD inhibitor into thesuitable culture medium of the competent plant cells. The competentcells are then transformed with the heterologous gene and the selectionmarker, and the transformed cells comprising the heterologous gene aregrown in a suitable medium and transformants selected therefrom. Thetransformed cells can then be regenerated into a fertile transformedplant.

J. Plants and Plant Parts

By “plant” is intended whole plants, plant organs (e.g., leaves, stems,roots, etc.), seeds, plant cells, propagules, embryos and progeny of thesame. Plant cells can be differentiated or undifferentiated (e.g.,callus, suspension culture cells, protoplasts, leaf cells, root cells,phloem cells, pollen). The present invention may be used forintroduction of polynucleotides into any plant species, including, butnot limited to, monocots and dicots. Examples of plants of interestinclude, but are not limited to, corn (maize), sorghum, wheat,sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean,sugar beet, sugarcane, tobacco, barley, and oilseed rape, Brassica sp.,alfalfa, rye, millet, safflower, peanuts, sweet potato, cassava, coffee,coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig,guava, mango, olive, papaya, cashew, macadamia, almond, oats,vegetables, ornamentals, and conifers.

Vegetables include, but are not limited to tomatoes, lettuce, greenbeans, lima beans, peas, and members of the genus Curcumis such ascucumber, cantaloupe, and musk melon. Ornamentals include, but are notlimited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils,petunias, carnation, poinsettia, and chrysanthemum. Crop plants are alsoof interest, including, for example, maize, sorghum, wheat, sunflower,tomato, crucifers, peppers, potato, cotton, rice, soybean, sugar beet,sugarcane, tobacco, barley, oilseed rape, etc.

This invention is suitable for any member of the monocot plant familyincluding, but not limited to, maize, rice, barley, oats, wheat,sorghum, rye, sugarcane, pineapple, yams, onion, banana, coconut, anddates.

K. Methods for Increasing Plant Yield

Methods for increasing plant yield are provided. The methods compriseproviding a plant comprising, or introducing into a plant or plant cell,a polynucleotide comprising a nucleotide sequence encoding an HPPD ofthe invention, growing the plant or a seed thereof in a field, andproducing a harvest from said plants or seeds. As defined herein, the“yield” of the plant refers to the quality and/or quantity of biomassproduced by the plant. By “biomass” is intended any measured plantproduct. An increase in biomass production is any improvement in theyield of the measured plant product. Increasing plant yield has severalcommercial applications. For example, increasing plant leaf biomass mayincrease the yield of leafy vegetables for human or animal consumption.Additionally, increasing leaf biomass can be used to increase productionof plant-derived pharmaceutical or industrial products. An increase inyield can comprise any statistically significant increase including, butnot limited to, at least a 1% increase, at least a 3% increase, at leasta 5% increase, at least a 10% increase, at least a 20% increase, atleast a 30%, at least a 50%, at least a 70%, at least a 100% or agreater increase.

In specific methods, the plant comprising an HPPD sequence of theinvention is treated with an effective concentration of an HPPDinhibitor herbicide, such as one or more HPPD inhibitor herbicide(s)selected from the group consisting of HPPD inhibitor herbicides of theclass of triketones (preferably benzobicyclon, sulcotrione, mesotrione,tembotrione, tefuryltrione, bicyclopyrone, fenquinotrione),diketonitriles, isoxazoles (preferably isoxaflutole), hydroxypyrazoles(preferably pyrazoxyfen, benzofenap, pyrazolynate, pyrasulfotole,topramezone, tolpyralate), N-(1,2,5-oxadiazol-3-yl)benzamides,N-(1,3,4-oxadiazol-2-yl)benzamides (preferably2-methyl-N-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide,N-(tetrazol-5-yl)- or N-(triazol-5-yl)arylcarboxamides (preferably2-chloro-3-ethoxy-4-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide,4-(difluoromethyl)-2-methoxy-3-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide,2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide(hereinafter also named “Cmpd. 1”),2-(methoxymethyl)-3-(methylsulfinyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide,pyridazinone derivatives, oxoprazine derivatives,N-(triazol-2-yl)arylcarboxamides, triazinones, and pyrazolones, wherethe herbicide application results in enhanced plant yield.

Methods for conferring herbicide tolerance in a plant or plant part arealso provided. In such methods, a nucleotide sequence encoding an HPPDof the invention is introduced into the plant, wherein expression of thepolynucleotide results in HPPD inhibitor herbicide tolerance. Plantsproduced via this method can be treated with an effective concentrationof an herbicide (such as one or more HPPD inhibitor herbicide(s)selected from the group consisting of HPPD inhibitor herbicides of theclass of triketones (preferably benzobicyclon, sulcotrione, mesotrione,tembotrione, tefuryltrione, bicyclopyrone, fenquinotrione),diketonitriles, isoxazoles (preferably isoxaflutole), hydroxypyrazoles(preferably pyrazoxyfen, benzofenap, pyrazolynate, pyrasulfotole,topramezone, tolpyralate), N-(1,2,5-oxadiazol-3-yl)benzamides,N-(1,3,4-oxadiazol-2-yl)benzamides (preferably2-methyl-N-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide,N-(tetrazol-5-yl)- or N-(triazol-5-yl)arylcarboxamides (preferably2-chloro-3-ethoxy-4-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide,4-(difluoromethyl)-2-methoxy-3-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide,2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide(hereinafter also named “Cmpd. 1”),2-(methoxymethyl)-3-(methylsulfinyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide,pyridazinone derivatives oxoprazine derivatives,N-(triazol-2-yl)arylcarboxamides, triazinones, and pyrazolones anddisplay an increased tolerance to the herbicide. An “effectiveconcentration” of an herbicide in this application is an amountsufficient to slow or stop the growth of plants or plant parts that arenot naturally tolerant or rendered tolerant to the herbicide.

L. Methods of Controlling Weeds in Afield

The present invention therefore also relates to a method of controllingundesired plants or for regulating the growth of plants in crops ofplants comprising a nucleotide sequence encoding an HPPD polypeptideaccording to the invention, where one or more HPPD inhibitor herbicides,for example, one or more HPPD inhibitor herbicides selected from theclass of triketones (preferably benzobicyclon, sulcotrione, mesotrione,tembotrione, tefuryltrione, bicyclopyrone, fenquinotrione),diketonitriles, isoxazoles (preferably isoxaflutole), hydroxypyrazoles(preferably pyrazoxyfen, benzofenap, pyrazolynate, pyrasulfotole,topramezone, tolpyralate), N-(1,2,5-oxadiazol-3-yl)benzamides,N-(1,3,4-oxadiazol-2-yl)benzamides (preferably2-methyl-N-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide,N-(tetrazol-5-yl)- or N-(triazol-5-yl)arylcarboxamides (preferably2-chloro-3-ethoxy-4-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide,4-(difluoromethyl)-2-methoxy-3-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide,2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide(hereinafter also named “Cmpd. 1”),2-(methoxymethyl)-3-(methylsulfinyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide-pyridazinonederivatives, oxoprazine derivatives, N-(triazol-2-yl)arylcarboxamides,triazinones, and pyrazolones, are applied to the plants (for exampleharmful plants such as monocotyledonous or dicotyledonous weeds orundesired crop plants), to the seeds (for example grains, seeds orvegetative propagules such as tubers or shoot parts with buds) or to thearea on which the plants grow (for example the area under cultivation).In this context, an effective concentration of one or more HPPDinhibitor herbicide(s), for example, one or more HPPD inhibitorherbicides selected from the group consisting of HPPD inhibitorherbicides of the class of triketones (preferably benzobicyclon,sulcotrione, mesotrione, tembotrione, tefuryltrione, bicyclopyrone,fenquinotrione), diketonitriles, isoxazoles (preferably isoxaflutole),hydroxypyrazoles (preferably pyrazoxyfen, benzofenap, pyrazolynate,pyrasulfotole, topramezone, tolpyralate),N-(1,2,5-oxadiazol-3-yl)benzamides, N-(1,3,4-oxadiazol-2-yl)benzamides(preferably2-methyl-N-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)-benzamide,N-(tetrazol-5-yl)- or N-(triazol-5-yl)arylcarboxamides (preferably2-chloro-3-ethoxy-4-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide,4-(difluoromethyl)-2-methoxy-3-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide,2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide(hereinafter also named “Cmpd. 1”),2-(methoxymethyl)-3-(methylsulfinyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide,pyridazinone derivatives, oxoprazine derivatives,N-(triazol-2-yl)arylcarboxamides, triazinones, and pyrazolones, can beapplied for example pre-planting (if appropriate also by incorporationinto the soil), pre-emergence or post-emergence, and may be combinedwith the application of other herbicides to which the crop is naturallytolerant, or to which it is resistant via expression of one or moreother herbicide resistance transgenes. See, e.g., U.S. App. Pub. No.2004/0058427 and PCT App. Pub. No. WO98/20144. By “effectiveconcentration” is intended the concentration which controls the growthor spread of weeds or other untransformed plants without significantlyaffecting the HPPD inhibitor-tolerant plant or plant seed. Those ofskill in the art understand that application of herbicides can take manydifferent forms and can take place at many different times prior toand/or throughout the seed planting and growth process. “Pre-emergent”application refers to an herbicide which is applied to an area ofinterest (e.g., a field or area of cultivation) before a plant emergesvisibly from the soil. “Post-emergent” application refers to anherbicide which is applied to an area after a plant emerges visibly fromthe soil. In some instances, the terms “pre-emergent” and“post-emergent” are used with reference to a weed in an area ofinterest, and in some instances these terms are used with reference to acrop plant in an area of interest. When used with reference to a weed,these terms may apply to a particular type of weed or species of weedthat is present or believed to be present in the area of interest.“Pre-plant incorporation” of an herbicide involves the incorporation ofcompounds into the soil prior to planting.

Thus, the present invention comprises a method of controlling weeds in afield comprising planting in a field a plant or a seed thereofcomprising an HPPD polypeptide of present invention and applying to saidplant or area surrounding said plant an effective concentration of oneor more HPPD inhibitor herbicides.

In one embodiment of this invention, a field to be planted with plants(such as soybean, cotton, corn, or wheat plants, e.g.) containing anHPPD nucleotide sequence of the invention, can be treated with an HPPDinhibitor herbicide, such as isoxaflutole (IFT), before the plants areplanted or the seeds are sown, which cleans the field of weeds that arekilled by the HPPD inhibitor herbicide, allowing for no-till practices,followed by planting or sowing of the plants in that same pre-treatedfield later on (burndown application using an HPPD inhibitor herbicide).The residual activity of IFT will also protect the emerging and growingplants from competition by weeds in the early growth stages. Once theplants have a certain size, and weeds tend to re-appear, glufosinate orglyphosate, or an HPPD inhibitor herbicide or a mixture of an HPPDinhibitor herbicide with another herbicide such as glyphosate, can beapplied as post-emergent herbicide over the top of the plants, when suchplants are tolerant to said herbicides.

In another embodiment of this invention, a field in which seedscontaining an HPPD nucleotide sequence of the invention were sown, canbe treated with an HPPD inhibitor herbicide, such as IFT, before theplants emerge but after the seeds are sown (the field can be madeweed-free before sowing using other means, typically conventionaltillage practices such as ploughing, chissel ploughing, or seed bedpreparation), where residual activity will keep the field free of weedskilled by the herbicide so that the emerging and growing plants have nocompetition by weeds (pre-emergence application of an HPPD inhibitorherbicide). Once the plants have a certain size, and weeds tend tore-appear, glufosinate or glyphosate, or an HPPD inhibitor herbicide ora mixture of an HPPD inhibitor herbicide with another herbicide such asglyphosate, can be applied as post-emergent herbicide over the top ofthe plants, when such plants are tolerant to said herbicides.

In another embodiment of this invention, plants containing an HPPDnucleotide sequence of the invention, can be treated with an HPPDinhibitor herbicide, over the top of the plants that have emerged fromthe seeds that were sown, which cleans the field of weeds killed by theHPPD inhibitor herbicide, which application can be together with (e.g.,in a spray tank mix), followed by or preceded by a treatment withglyphosate or glufosinate as post-emergent herbicide over the top of theplants (post-emergence application of an HPPD inhibitor herbicide (withor without glyphosate)), when such plants are tolerant to suchherbicides.

Examples of individual representatives of the monocotyledonous anddicotyledonous weeds which can be controlled with an HPPD inhibitorherbicide include:

-   -   Monocotyledonous harmful plants of the genera: Aegilops,        Agropyron, Agrostis, Alopecurus, Apera, Avena, Brachiaria,        Bromus, Cenchrus, Commelina, Cynodon, Cyperus, Dactyloctenium,        Digitaria, Echinochloa, Eleocharis, Eleusine, Eragrostis,        Eriochloa, Festuca, Fimbristylis, Heteranthera, Imperata,        Ischaemum, Leptochloa, Lolium, Monochoria, Panicum, Paspalum,        Phalaris, Phleum, Poa, Rottboellia, Sagittaria, Scirpus,        Setaria, Sorghum.    -   Dicotyledonous weeds of the genera: Abutilon, Amaranthus,        Ambrosia, Anoda, Anthemis, Aphanes, Artemisia, Atriplex, Bellis,        Bidens, Capsella, Carduus, Cassia, Centaurea, Chenopodium,        Cirsium, Convolvulus, Datura, Desmodium, Emex, Erysimum,        Euphorbia, Galeopsis, Galinsoga, Galium, Hibiscus, Ipomoea,        Kochia, Lamium, Lepidium, Lindernia, Matricaria, Mentha,        Mercurialis, Mullugo, Myosotis, Papaver, Pharbitis, Plantago,        Polygonum, Portulaca, Ranunculus, Raphanus, Rorippa, Rotala,        Rumex, Salsola, Senecio, Sesbania, Sida, Sinapis, Solanum,        Sonchus, Sphenoclea, Stellaria, Taraxacum, Thlaspi, Trifolium,        Urtica, Veronica, Viola, Xanthium.

HPPD inhibitor herbicides useful in the present invention, including butnot limited to HPPD inhibitor herbicides of the class of triketones(preferably benzobicyclon, sulcotrione, mesotrione, tembotrione,tefuryltrione, bicyclopyrone, fenquinotrione), diketonitriles,isoxazoles (preferably isoxaflutole), hydroxypyrazoles (preferablypyrazoxyfen, benzofenap, pyrazolynate, pyrasulfotole, topramezone,tolpyralate), N-(1,2,5-oxadiazol-3-yl)benzamides,N-(1,3,4-oxadiazol-2-yl)benzamides (preferably2-methyl-N-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)-benzamide,N-(tetrazol-5-yl)- or N-(triazol-5-yl)arylcarboxamides (preferably2-chloro-3-ethoxy-4-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide,4-(difluoromethyl)-2-methoxy-3-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide,2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide(hereinafter also named “Cmpd. 1”),2-(methoxy-methyl)-3-(methylsulfinyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide,pyridazinone derivatives, oxoprazine derivatives,N-(triazol-2-yl)arylcarboxamides, triazinones, and pyrazolones, can beformulated in various ways, depending on the prevailing biologicaland/or physico-chemical parameters. Examples of possible formulationsare: wettable powders (WP), water-soluble powders (SP), water-solubleconcentrates, emulsifiable concentrates (EC), emulsions (EW), such asoil-in-water and water-in-oil emulsions, sprayable solutions, suspensionconcentrates (SC), oil- or water-based dispersions, oil-misciblesolutions, capsule suspensions (CS), dusts (DP), seed-dressing products,granules for application by broadcasting and on the soil, granules (GR)in the form of microgranules, spray granules, coated granules andadsorption granules, water-dispersible granules (WG), water-solublegranules (SG), ULV formulations, microcapsules and waxes.

These individual types of formulation are known in principle and aredescribed, for example, in: Winnacker-Kiichler, “Chemische Technologie”[Chemical technology], Volume 7, C. Hanser Verlag Munich, 4th Ed. 1986;Wade van Valkenburg, “Pesticide Formulations”, Marcel Dekker, N.Y.,1973; K. Martens, “Spray Drying” Handbook, 3rd Ed. 1979, G. Goodwin Ltd.London.

The formulation auxiliaries required, such as inert materials,surfactants, solvents and further additives, are also known and aredescribed, for example, in: Watkins, “Handbook of Insecticide DustDiluents and Carriers”, 2nd Ed., Darland Books, Caldwell N.J., H.v.Olphen, “Introduction to Clay Colloid Chemistry”; 2nd Ed., J. Wiley &Sons, N.Y.; C. Marsden, “Solvents Guide”; 2nd Ed., Interscience, N.Y.1963; McCutcheon's “Detergents and Emulsifiers Annual”, MC Publ. Corp.,Ridgewood N.J.; Sisley and Wood, “Encyclopedia of Surface ActiveAgents”, Chem. Publ. Co. Inc., N.Y. 1964; Schönfeldt,“Grenzflächenaktive Äthylenoxidaddukte” [Interface-active ethylene oxideadducts], Wiss. Verlagsgesell., Stuttgart 1976; Winnacker-Kiichler,“Chemische Technologie” [Chemical technology], Volume 7, C. HanserVerlag Munich, 4th Ed. 1986.

Based on these formulations, it is also possible to prepare combinationswith other pesticidally active substances such as, for example,insecticides, acaricides, herbicides, fungicides, and with safeners,fertilizers and/or growth regulators, for example in the form of a readymix or a tank mix.

M. Methods of Introducing Gene of the Invention into Another Plant

Also provided herein are methods of introducing the HPPD nucleotidesequence of the invention into another plant. The HPPD nucleotidesequence of the invention, or a fragment thereof, can be introduced intosecond plant by recurrent selection, backcrossing, pedigree breeding,line selection, mass selection, mutation breeding and/or genetic markerenhanced selection.

Thus, in one embodiment, the methods of the invention comprise crossinga first plant comprising an HPPD nucleotide sequence of the inventionwith a second plant to produce F1 progeny plants and selecting F1progeny plants that are tolerant to an HPPD inhibitor herbicide or thatcomprise the HPPD nucleotide sequence of the invention. The methods mayfurther comprise crossing the selected progeny plants with the firstplant comprising the HPPD nucleotide sequence of the invention toproduce backcross progeny plants and selecting backcross progeny plantsthat are tolerant to an HPPD inhibitor herbicide or that comprise theHPPD nucleotide sequence of the invention. Methods for evaluating HPPDinhibitor herbicide tolerance are provided elsewhere herein. The methodsmay further comprise repeating these steps one or more times insuccession to produce selected second or higher backcross progeny plantsthat are tolerant to an HPPD inhibitor herbicide or that comprise theHPPD nucleotide sequence of the invention.

Any breeding method involving selection of plants for the desiredphenotype can be used in the method of the present invention. In someembodiments, the F1 plants may be self-pollinated to produce asegregating F2 generation. Individual plants may then be selected whichrepresent the desired phenotype (e.g., HPPD inhibitor herbicidetolerance) in each generation (F3, F4, F5, etc.) until the traits arehomozygous or fixed within a breeding population.

The second plant can be a plant having a desired trait, such asherbicide tolerance, insect tolerance, drought tolerance, nematodecontrol, water use efficiency, nitrogen use efficiency, improvednutritional value, disease resistance, improved photosynthesis, improvedfiber quality, stress tolerance, improved reproduction, and the like.The second plant may be an elite event as described elsewhere herein.

In various embodiments, plant parts (whole plants, plant organs (e.g.,leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos,and the like) can be harvested from the resulting cross and eitherpropagated or collected for downstream use (such as food, feed, biofuel,oil, flour, meal, etc).

N. Methods of Obtaining a Plant Product

The present invention also relates to a process for obtaining acommodity product, comprising harvesting and/or milling the grains froma crop comprising an HPPD sequence of the invention to obtain thecommodity product. Agronomically and commercially important productsand/or compositions of matter including but not limited to animal feed,commodities, and plant products and by-products that are intended foruse as food for human consumption or for use in compositions andcommodities that are intended for human consumption, particularlydevitalized seed/grain products, including a (semi-)processed productsproduced from such grain/seeds, wherein said product is or compriseswhole or processed seeds or grain, animal feed, corn or soy meal, cornor soy flour, corn, corn starch, soybean meal, soy flour, flakes, soyprotein concentrate, soy protein isolates, texturized soy proteinconcentrate, cosmetics, hair care products, soy nut butter, natto,tempeh, hydrolyzed soy protein, whipped topping, shortening, lecithin,edible whole soybeans (raw, roasted, or as edamame), soy yogurt, soycheese, tofu, yuba, as well as cooked, polished, steamed, baked orparboiled grain, and the like are intended to be within the scope of thepresent invention if these products and compositions of matter containdetectable amounts of the nucleotide and/or amino acid sequences setforth herein as being diagnostic for any plant containing suchnucleotide sequences.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Overview

Example 1: Creation of mutated HPPD polypeptides by site-directedmutagenesis

Example 2: Cloning, expression, and purification of recombinantwild-type and mutant HPPD polypeptides

Example 3: HPPD enzyme assay to analyse mutant HPPD polypeptides withimproved HPPD inhibitor herbicide tolerance

Example 4: Improved herbicide tolerance mediated by residue exchanges inHPPD polypeptides

Example 5: Soybean transformation and tolerance of the T0 soybean plants

Example 6: Cotton T0 plant establishment and selection

Example 7: Transformation of Maize Plant Cells by Agrobacterium-MediatedTransformation

Example 1. Creation of Mutated HPPD Polypeptides by Site-DirectedMutagenesis

The Pseudomonas fluorescens HPPD nucleotide sequence (SEQ ID NO:72) asdescribed in WO2009/144079 encoding the HPPD polypeptide correspondingto SEQ ID NO:1 was cloned according to well known methods in the art andas described in WO2014/043435. Subsequent site-saturated mutagenesis,site-directed mutagenesis and combinatorial variants with one or moremutations of the nucleic acid encoding sequence of wild-type PfHPPDpolypeptide encoding the recombinant HPPD polypeptide corresponding toSEQ ID NO: 1 were carried out using standard PCR-based technologies wellknown in the art (and as described likewise in WO2014/043435). Alldesigned and synthesized mutant clones were confirmed by DNA sequencingusing plasmid specific oligonucleotides. Table 2, below, summarizes theexemplary mutant HPPD polypeptides (SEQ ID NO:2 to NO:71).

TABLE 2 Overview of exemplary amino acid exchangescorresponding to amino acidposition in SEQ ID NO: 1. SEQAmino acid position relative to HPPD ID polypeptide SEQ ID NO: 1 NO: 213215 268 270 315 335 336 337 339 340 344 345  1 R P P T T E G N K A S I 2 P W A Q  3 A P D S V  4 G P D S V  5 P D S V  6 A N P D S V  7 G N PD S V  8 N P D S V  9 A P D S V V 10 G P D S V V 11 P D S V V 12 A N P DS V V 13 G N P D S V V 14 N P D S V V 15 R N P D S V V 16 S N P D S V V17 A E P D S V V 18 E P D S V V 19 R E P D S V V 20 S E P D S V V 21 A PD S V Q V 22 G P D S V Q V 23 P D S V Q V 24 A P H S V 25 G P H S V 26 PH S V 27 A N P H S V 28 G N P H S V 29 N P H S V 30 A P H S V V 31 G P HS V V 32 P H S V V 33 A N P H S V V 34 G N P H S V V 35 N P H S V V 36 RN P H S V V 37 S N P H S V V 38 A E P H S V V 39 G E P H S V V 40 G E PD S V V 41 R E P H S V V 42 S E P H S V V 43 A E P H S V Q V 44 G E P HS V Q V 45 A N P H S V Q V 46 G N P H S V Q V 47 A N P H S V Q M 48 G NP H S V Q M 49 N P H S V Q M 50 S N P H S V Q M 51 A N P D S V K 52 A NP D S V Q 53 A N P D S V Q V 54 A N P D S V Q K 55 A N P H S V K 56 A NP H S V Q 57 A N P H S V Q K 58 A S P D S V 59 A S P D S V Q K 60 A S PD S V K 61 A S P H S V 62 A S P H S V M 63 K A A S P H S V Q 64 A S P HS V Q M 65 A S R P H S V M 66 K A S P H S V Q M 67 K A S R P H S V Q M68 A S Q P H S V Q M 69 A S R P H S V Q M 70 K A S Q P H S V Q M 71 L PF E

For clarity, empty cells at the respective amino acid position in SEQ IDNO:2 to NO:71 are defined as identical to the amino acids correspondingto SEQ ID NO:1, highlighting only the exchanges in the mutant HPPDpolypeptides. The mutant HPPD polypeptides represented here are examplesby a way of illustration, not by a way of limitation.

Example 2: Cloning, Expression, and Purification of RecombinantWild-Type and Mutant HPPD Polypeptides

All resulting nucleic acid encoding sequences of wild-type and mutantHPPD encoding the recombinant HPPD polypeptide were cloned, produced andpurified using methods well known in the art (Sambrook et al., MolecularCloning: A Laboratory Manual, 3rd ed., CSH Laboratory Press, 2001, ColdSpring Harbor, N.Y.). All resulting nucleic acid encoding sequences werecloned into pSE420(RI)NX fused with an N-terminal His-tag (encoding theamino acid sequence M1-A2-H3-H4-H5-H6-H7-H8-), as described inWO2014/043435, and were expressed in Escherichia coli strain BL21 (DE3)(New England Biolabs, Frankfurt, Germany). For clarity, all listedpositions with the respective amino acid exchanges from mutant HPPDpolypeptides in Tables 1 to 5 corresponding to SEQ ID NO:2 to NO:71 inthis invention, refer to the native wild-type HPPD amino acid sequencewithout the N-terminal His-tag corresponding to SEQ ID NO:1.

For the generation of purified HPPD polypeptide samples, cells weregrown for 3 h at 37° C. in 5 ml LB medium containing 100 μg/mlampicillin in a 50 ml shaker flask at 140 rpm. One ml of this starterculture was used as inoculum for the expression culture. Cells weregrown for about 3 h at 37° C. in 100 ml LB medium containing 100 μg/mlampicillin and 150 mM Hepes (Merck, Darmstadt, Germany) in a 500 mlshaker flask at 120 rpm. At an OD600 of about 0.6, IPTG (Roth,Karlsruhe, Germany) was added to a concentration of 0.4 mM. Afterfurther growth for 60 min at 37° C., the temperature was reduced to 28°C. and growth continued for another 18 h at 140 rpm. Cells wereharvested by centrifugation at 4° C., 3200 g for 30 min in 50 ml Falcontubes and cell pellets were stored at −80° C. Cells were lysed andhis-tagged protein was purified according to manufacturer protocol ofthe used Ni-NTA Fast Start Kit (Qiagen, Hilden, Germany) with followingadaptions for increased yield: cells from 50 ml culture were lysed in 4ml and lysate supernatant was generated by centrifugation for 15 min at18000 g. The amount of matrix in the columns was increased by additionof 1 ml of NiNTA Superflow (Qiagen, Hilden, Germany) each andextensively re-buffered into 20 mM Tris (pH 7.6) (Merck, Darmstadt,Germany). Lysate supernatant was applied and His-tagged protein wasbound to the Ni-NTA matrix by incubation for 1 h at 4° C.

The resulting protein samples were re-buffered into 20 mM Tris, 20%Glycerol (pH 7.8) (Sigma-Aldrich, St. Louis, USA) by use of Zeba™ SpinDesalting Columns, 7K MWCO, 10 mL (Thermo Fisher Scientific, Waltham,USA) and analysed for protein concentration and purity by Abs280(NANODROP 8000, Thermo Fisher Scientific, Waltham, USA) and SDS-PAGE.The concentrations of purified proteins were generally in the range of0.6-4.6 mg/ml by an estimated purity of about 90%. For the generation ofcrude HPPD polypeptide extract in micro titer plates (MTP) for thedetermination of residual activity in inhibition assays, cells weregrown in 40 or 150 μl LB medium containing 1% Glucose (Merck, Darmstadt,Germany) and 100 μg/ml ampicillin in a standard 96 well plate (ThermoFisher Scientific, Waltham, USA) incubated for about 18 h in a humidityincubator at 37° C. 30 μl of this starter culture were added to 600 μlLB medium containing 100 μg/ml ampicillin and 150 mM Hepes (Merck,Darmstadt, Germany) as inoculum for the expression culture in 96 wellplates (2 ml deep wells; HJ Bioanalytik, Erkelenz, Germany). The plateswere sealed by an aluminium foil, and cells were incubated for 5 h at37° C. on a plate shaker at 750 rpm. The expression was induced byaddition of IPTG in a final concentration of 1 mM followed by furthersealed incubation for about 18 h at 30° C. on a plate shaker at 750 rpm.

Cells were harvested by centrifugation at 4° C., 2500 g for 15 mindiscarding the supernatant. Cell pellets were stored at −80° C. andlysed in 250 μl 1× BugBuster® (Merck, Darmstadt, Germany) in 140 mM Tris(pH 7.8), with 1:25000 diluted BNase® (Qiagen, Hilden, Germany) byincubation of the resuspended cells for 30 min at 4° C. and 1000 rpm.Lysates were clarified by centrifugation for 15 min at 4° C., 2500 g,and 150 μl supernatant were transferred in standard 96 well plate(Thermo Fisher Scientific, Waltham, USA) for subsequent testing inquadruplets.

Example 3: HPPD Enzyme Assay to Analyse Mutant HPPD Polypeptides withImproved HPPD Inhibitor Herbicide Tolerance

The activity of HPPD polypeptides was determined in absence or presenceof HPPD inhibitors using the coupled HPPD activity assay (FIG. 1).

For the determination of the residual activity, the apparent kineticconstant (k_(app)) of the determined substrate conversion was measuredas kinetic changes in absorbance (OD) at 320 nm in a coupled assay, inthat homogentisate (HGA) formed by HPPD from HPP is directly convertedinto the well absorbing molecule maleylacetoacetate (MAA) by a secondenzyme homogentisate dioxygenase (HGD), applied in excess uniformly inall assays (see FIG. 1). The measurements were performed in 384 microtiter plates (Greiner Bio-One GmbH, Frickenhausen, Germany) by platereaders (Tecan infinite M1000 or M1000PRO, Tecan, Männedorf,Switzerland). The k_(cat)/k_(M) ratio of an enzymatic activity isproportional to the apparent kinetic constant k_(app) and isproportional to k_(cat)/k_(M)*[E] ([E]=enzyme concentration). Acompetitive inhibitor exhibits an apparent increase in k_(M) and therebya reciprocal decrease in k_(app) at non-saturating substrateconcentrations. As both k_(app) measurements in the presence and absenceof inhibitor were performed by use of the identical enzyme sample, crudeor purified, and thereby at the same enzyme concentration, the enzymeconcentration eliminates from the calculation of residual activity andthe ratio of both k_(app) directly indicates the change of k_(M) due tothe inhibition. Noteworthy, this concept applies to enzyme/inhibitorpairs interacting in a “competitive inhibition” manner, probably correctfor almost all polypeptide variants and inhibitors described herein, butfor sure not with respect to the wild-type polypeptide, which isinhibited irreversibly (for comparison see WO2014/043435, FIG. 2).Consequently, residual activities of the wild-type HPPD polypeptidereferring to “competitive inhibition” and ki values can't be correctlycalculated.

The assay solution used for determination of residual activities in rawHPPD polypeptide samples was composed by 200 mM sodium phosphate (Merck,Darmstadt, Germany, pH 7.0), 5 mM MgCl₂ (Merck, Darmstadt, Germany), 20mM ascorbate (Sigma-Aldrich, St. Louis, USA), 1 mM DTT (Sigma-Aldrich,St. Louis, USA), 0.1% Pluronic F-68 (Sigma-Aldrich, St. Louis, USA), 40μM FeSO₄ (Sigma-Aldrich, St. Louis, USA), about 8 mg/ml purified HGD and500 μM HPP from a 1 M stock solution in DMSO (Sigma-Aldrich, St. Louis,USA) and equilibrated for 20 min on ice. For every HPPD polypeptidesample two assays were performed in quadruplets, whereby 5 μl of HPPDpolypeptide sample were mixed firstly with 5 μl buffer (1×BugBuster®;(Merck, Darmstadt, Germany); in 140 mM Tris, pH 7.8, with 1:25000diluted BNase®; Qiagen, Hilden, Germany)) or 5 μl inhibitor diluted inthe same buffer from a 0.1 M stock solution in DMSO (200 μM resulting in100 μM in the HPPD polypeptide/inhibitor sample) in the reference andinhibition assay, respectively, and subsequently with 10l assaysolution. The change in absorbance at 320 nm was followed in 1 minintervals for 30 min. The k_(app) values were calculated as signal slopeover time in the early phase of the kinetic reaction, usually for thefirst 5-10 minutes of the measurements. Additionally and according tothe calculated residual activity, the total conversion, i.e. theabsolute change in signal, in the 30 min timeframe was monitored asmeasure of turnover, and a residual turnover was calculated by dividingthe change in signal in the presence of inhibitor by the change insignal in the reference sample without inhibitor.

The assay solutions used for determination of ki values were composed inthe same way containing six different concentrations of HPP substrate(0-1350 μM) for each of the four inhibitor concentrations tested. Theinhibitors were diluted in 140 mM Tris, 0.05% Pluronic F-68(Sigma-Aldrich, St. Louis, USA) and applied in concentrations adoptedfor the respective HPPD polypeptide/inhibitor pairs to generate dynamicdata; generally, their concentrations in the HPPD polypeptide/inhibitorsample were in the range from 0 to 0.001 M.

Example 4: Improved Herbicide Tolerance Mediated by Residue Exchanges inHPPD Polypeptides

When the tolerance of mutant HPPD polypeptides was determined againstHPPD inhibitor herbicide, it became evident that some of the newembodiments in this invention are not only significantly improvedcompared to reference wild-type HPPD (SEQ ID NO:1), but alsounexpectedly better than the prior art mutant HPPD polypeptides (like,for example, those being disclosed in WO2014/043435) with SEQ ID NO:2 inthis invention as an example.

As outlined in Table 3, prior art mutant HPPD polypeptides(WO2014/043435) corresponding to SEQ ID NO:2 in this invention containsresidue exchanges at position 335, 336, 339 and 340.

Based on mutant HPPD polypeptide comprising 268 (P=>A), 335 (E=>P), 336(G=>D/H), 337 (N=>S), the introduction of further residue exchanges atposition 213, 215, 270, 315, 340, 344, and/or 345 generated mutant HPPDpolypeptides showing exemplary strongly improved tolerance (Table 3),concerning the applied HPPD inhibitor.

Accordingly, we generated and evaluated new mutant HPPD polypeptides bycombinatorial residue exchanges at position 268 (proline=>alanine), 335(glutamic acid=>proline), 336 (glycine=>aspartic acid/histidine), 337(asparagine=>serine) and, optionally, further comprising exchanges atposition 213, 215, 270, 315, 340, 344, and/or 345 (Table 3 and 4), thatexhibit improved turnover, higher residual turnover, and higher kivalues and thereby significantly higher herbicide tolerance. The levelof improvement might differ concerning the HPPD polypeptides employed insuch assay, with a level of up to 25 fold, compared to SEQ ID NO: 2(Table 4).

Analysis of the time-course of inhibition against the HPPD inhibitorherbicide chemical class revealed, that the HPPD inhibitor herbicidesappear to be reversible inhibitors against the new mutant HPPDpolypeptides, in contrast to the slow and tight binding inhibitorcharacteristic of the wild-type HPPD polypeptide corresponding to SEQ IDNO:1 (see FIG. 2). These behaviors provide a better and versatilepotential for tolerances in crop plants to HPPD inhibitor herbicides.

For high residual activity in the presence of HPPD inhibitor herbicides,the disclosed positions and residue changes are highlighted in Table 1relative to the amino acid position in the HPPD polypeptidecorresponding to SEQ ID NO:1 in this invention are shown to beimportant.

TABLE 3Tolerance of mutant HPPD polypeptides against HPPD inhibitor herbicides.Turnover in the absence of Cmpd. 1 and residual turnover in the presence of Cmpd. 1according to Example 3 at high substrate concentration and 100 μM inhibitor. Turnover was measured as kinetic changes in absorbance (OD) at 320 nm in thecoupled assay. SEQ Residual ID Residual turnover NO: 213 215 268 270 315335 336 337 339 340 344 345 Turnover turnover [%]  1 R P P T T E G N K AS I 0.203 0.021  2 P W A Q 0.060 0.026  3 A P D S V 0.096 0.061 64%  4 GP D S V 0.126 0.055 44%  5 P D S V 0.157 0.059 38%  6 A N P D S V 0.1650.082 50%  7 G N P D S V 0.149 0.063 42%  8 N P D S V 0.107 0.054 50%  9A P D S V V 0.102 0.053 52% 10 G P D S V V 0.125 0.070 56% 11 P D S V V0.160 0.071 44% 12 A N P D S V V 0.115 0.058 50% 13 G N P D S V V 0.024n.d. 14 N P D S V V 0.188 0.062 33% 15 R N P D S V V 0.040 0.030 16 S NP D S V V 0.181 0.083 46% 17 A E P D S V V 0.138 0.081 59% 18 E P D S VV 0.134 0.057 43% 19 R E P D S V V 0.141 0.063 45% 20 S E P D S V V0.199 0.105 53% 21 A P D S V Q V 0.157 0.080 51% 22 G P D S V Q V 0.1320.062 47% 23 P D S V Q V 0.193 0.076 39% 24 A P H S V 0.271 0.105 39% 25G P H S V 0.215 0.083 39% 26 P H S V 0.279 0.077 28% 27 A N P H S V0.266 0.101 38% 28 G N P H S V 0.204 0.088 43% 29 N P H S V 0.289 0.08429% 30 A P H S V V 0.311 0.149 48% 31 G P H S V V 0.268 0.098 37% 32 P HS V V 0.326 0.128 39% 33 A N P H S V V 0.189 0.087 46% 34 G N P H S V V0.243 0.113 47% 35 N P H S V V 0.036 0.023 36 R N P H S V V 0.108 0.06661% 37 S N P H S V V 0.096 0.048 50% 38 A E P H S V V 0.286 0.129 45% 39G E P H S V V 0.244 0.097 40% 40 G E P D S V V 0.134 0.073 54% 41 R E PH S V V 0.280 0.105 38% 42 S E P H S V V 0.278 0.111 40% 43 A E P H S VQ V 0.190 0.110 58% 44 G E P H S V Q V 0.036 n.d. 45 A N P H S V Q V0.214 0.121 57% 46 G N P H S V Q V 0.179 0.098 55% 47 A N P H S V Q M0.314 0.166 53% 48 G N P H S V Q M 0.225 0.117 52% 49 N P H S V Q M0.316 0.103 33% 50 S N P H S V Q M 0.315 0.138 44% 51 A N P D S V K0.131 0.074 56% 52 A N P D S V Q 0.189 0.088 47% 53 A N P D S V Q V0.199 0.107 54% 54 A N P D S V Q K 0.089 0.059 66% 55 A N P H S V K0.189 0.095 50% 56 A N P H S V Q 0.152 0.086 57% 57 A N P H S V Q K0.167 0.099 59% 58 A S P D S V 0.144 0.065 45% 59 A S P D S V Q K 0.1310.074 56% 60 A S P D S V K 0.103 0.046 45% 61 A S P H S V 0.229 0.09140% 62 A S P H S V M 0.308 0.115 37% 63 K A A S P H S V Q 0.23 0.156 68%64 A S P H S V Q M 0.242 0.114 47% 65 A S R P H S V M 0.327 0.177 54% 66K A S P H S V Q M 0.336 0.187 56% 67 K A S R P H S V Q M 0.335 0.215 64%68 A S Q P H S V Q M 0.346 0.223 64% 69 A S R P H S V Q M 0.36 0.237 66%70 K A S Q P H S V Q M 0.338 0.240 71% 71 L P F E 0.044 0.022

Residual turnover were determined according to Example 3 by measuringtotal change in signal in the presence and absence of Cmpd. 1(2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide).For each mutant HPPD polypeptide, the total change in signal withoutHPPD inhibitor herbicides served for normalization of the total changein signal in the presence of the herbicide. The summarized “values forthe turnover” in the respective table are medians of measurements inquadruplets with an average standard deviation of 5%. For clarity, emptycells at the respective amino acid position in SEQ ID NO:2 to NO:71 aredefined as identical to the amino acids corresponding to SEQ ID NO: 1,highlighting only the exchanges in the HPPD polypeptide variant. At thegiven inhibitor concentration of 100 M Cmpd. 1 in the assay, thereference wild-type HPPD (SEQ ID NO:1), and also the prior art mutantHPPD polypeptide (like, for example, those being disclosed inWO2014/043435) with SEQ ID NO:2 or the mutant HPPD polypeptide (like,for example, those being disclosed in WO2015/135881) with SEQ ID NO:71in this invention as an example, do not exhibit any significant kineticchanges in absorbance at 320 nm (Abs320) in the coupled HPPD activityassay compared to the values measured with the knock-out HPPDpolypeptide. The value for the knock-out HPPD polypeptide withoutinhibitor was at 0.021 (changes in absorbance at 320 nm) and the valuesthe HPPD polypeptides corresponding to SEQ ID NO:1, SEQ ID NO:2, and SEQID NO:71 were 0.021, 0.026, and 0.022 at a inhibitor concentration of100 M, respectively.

The knock-out HPPD polypeptide was obtained by exchanging a histidine toan alanine at the amino acid position corresponding to amino acidposition 162 of SEQ ID NO: 1. This position is well known for itsimportance due to its involvement in the coordinated binding of the ironatom in the active site of the HPPD polypeptide (Serre et al. (1999),Structure, 7, 977-988).

The mutant HPPD polypeptide corresponding to SEQ ID NO:3 with amino acidexchanges at positions 268, 335, 336, 337, and 340 relative to HPPDpolypeptide according to SEQ ID NO:1, exhibit in the presence of theHPPD inhibitor tested an improvement regarding residual turnovers (Table3) vs. the wild-type HPPD polypeptide (SEQ ID NO:1), but also vs. priorart (WO2014/043435 and WO2015/135881) mutant HPPD polypeptidescorresponding to SEQ ID NO:2 and SEQ ID NO:71 in this invention. SEQ IDNO: 6, which differ only at the positions 270 from HPPD polypeptideaccording to SEQ ID NO:3, exhibit an increase in the absolute turnoverand absolute residual turnover in the presence of HPPD inhibitor Cmpd.1(Table 3).

Starting from SEQ ID NO: 26 and introducing an alanine at the amino acidposition 268 corresponding to SEQ ID NO:1, yielding SEQ ID NO: 24exhibits an increase in the residual turnover in the presence of HPPDinhibitor Cmpd.1 (Table 3).

Starting from SEQ ID NO: 49 with amino acid exchanges at positions 270,335, 336, 337, 340, 344 and 345 relative to HPPD polypeptide accordingto SEQ ID NO:1 and introducing an alanine at the amino acid position 268(SEQ ID NO: 47), exhibits a significant increase in the residualturnover in the presence of HPPD inhibitor Cmpd.1 (Table 3).

Introducing and combining above mentioned exchanges in the positions268, 270, 335, 336, 337, 340, 344, 345 demonstrating the importance ofthe combinatorial residue exchanges at the disclosed amino acidpositions.

Further improvements in HPPD inhibitor tolerance are apparent invariants with residue exchanges at the disclosed amino acid positions213, 215 and 315. Exemplary, mutant HPPD polypeptides SEQ ID NO: 66, SEQID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70 haveadditional combinatorial residue exchanges at position 213 and/or 315compared to mutant HPPD polypeptide SEQ ID NO:64. These changes increasesignificantly the turnover and the residual turnover in the presence ofHPPD inhibitor Cmpd.1 (Table 3).

TABLE 4 Evaluation of tolerance of mutated HPPD polypeptides against HPPD inhibitor herbicide Cmpd.1 by the determination of the ki valuesSEQ Amino acid position relative to HPPD polypeptide Cmpd. 1 IDSEQ ID NO: 1 ki NO: 213 215 268 270 315 335 336 337 339 340 344 345 [μM] 1 R P T T E G N K A S I —  2 P W A Q  1 47 A N P H S V Q M 25 63 K A AS P H S V Q 24 68 A S Q P H S V Q M 28 70 K A S Q P H S V Q M 22

For clarity, empty cells at the respective amino acid position in SEQ IDNO:2 to NO:71 are defined as identical to the amino acids correspondingto SEQ ID NO:1, highlighting only the exchanges in the mutant HPPDpolypeptides. The mutant HPPD polypeptides represented here are examplesby a way of illustration, not by a way of limitation.

Data were obtained by measuring the initial reaction rates withincreasing concentrations of Cmpd. 1(2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide).Generally, six different concentrations of HPP substrate (0-1350 μM) andfour different concentrations of the respective inhibitor were applied.The inhibitor concentrations were adopted for the respective HPPDpolypeptide/inhibitor pairs to generate dynamic data, i.e. variants withlower tolerance were analyzed in a range of lower inhibitorconcentrations, and concentrations of up to 1000 μM were used forvariants with maximized tolerance. GraphPad Prism (version 6.00 forWindows, GraphPad Software, La Jolla Calif. USA) were used for dataanalysis and fitting of kinetic constants applying constraints accordingto a competitive inhibition mode. Where obvious outliers occurred, oractivities obtained at very high substrate concentrations didn't obeythe mathematics underlying the competitive inhibition mode, respectivevalues were excluded from the fit.

Example 5: Soybean Transformation and Tolerance of the T0 Soybean Plants

Soybean transformation was achieved by using methods well known in theart, such as the one described using the Agrobacterium tumefaciensmediated transformation soybean half-seed explants using essentially themethod described by Paz et al. (2006), Plant cell Rep. 25:206.Transformants were identified by using the HPPD inhibitor herbicide“tembotrione” as selection marker. The appearance of green shoots wasobserved, and documented as an indicator of tolerance to the HPPDinhibitor herbicide tembotrione. The tolerant transgenic shoots showednormal greening comparable to wild-type soybean shoots not treated withHPPD inhibitor herbicide tembotrione, whereas wild-type soybean shootstreated with the same amount of HPPD inhibitor herbicide tembotrionewere entirely bleached. This indicated that the presence of therespective HPPD polypeptide enabled the tolerance to HPPD inhibitorherbicides, like tembotrione.

Tolerant green shoots were transferred to rooting media or grafted.Rooted plantlets were transferred to the greenhouse after an acclimationperiod. Plants containing the transgene were then sprayed with HPPDinhibitor herbicides, as for example with mesotrione at a rate of300-600 g AI/ha, or with Cmpd. 1(2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)-benzamide)at a rate of 150 g-300 g AI/ha supplemented with ammonium sulfate andmethyl ester rapeseed oil. Five to ten days after the application, thesymptoms due to the application of the herbicide were evaluated andcompared to the symptoms observed on wild-type plants under the sameconditions. For example, T0 soybean plants having a “plant expressioncassette”, which includes an HPPD inhibitor tolerant HPPD polypeptide ofthe present invention, were tested towards the tolerance of Cmpd. 1.Prior greenhouse trials with the transgenic plants, soybeantransformants were routinely analyzed for the expression and presence ofthe transgenes using the ELISA protein detection method (see detaileddescription under item D and H). Only plants recovering in the selectionmedia and having a detectable HPPD transgene protein expression wereused for the herbicide tolerance analysis. A DeVries Tracker Sprayer wascalibrated prior to each spraying. The chemical formulation used forCmpd. 1 was supplemented with ammonium sulfate and methylated rape seedoil. Spray tests were conducted with a concentration, which equals to300 grams AI per hectare (300 g AI/ha).

In greenhouse trials, independent T0 soybean events containing anexemplary mutant HPPD polypeptide were sprayed with the HPPD inhibitorherbicide Cmpd.1(2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide)at the rate of 300 grams AI/ha, supplemented with ammonium sulfate andmethyl ester rapeseed oil. Five days after the application, the leafdamaged area due to the HPPD inhibitor herbicide is scored in a scalefrom 0 (no damage) to 100 (complete bleaching). Under those conditions,the wild-type plants were completely bleached and their damage scoreswere in the 95-100 range.

Table 5 presents the distribution of the HPPD inhibitor herbicide damagescore data as percentile for exemplary mutant HPPD inhibitor herbicidetolerant polypeptides (SEQ ID NOs). The percentiles normalize ranks ofthe damage score from an individual plant in a population. The value ofthe 25th percentile is the damage score where 25% of the soybean eventsin the given population had a lower and 75% higher damage scores. Themedian is the 50th percentile. Half the values had higher damage scores;half had lower damage scores. The value of the 75th and 90th percentileis the damage score where 75% and 90% of the soybean events had lowerdamage scores, respectively. The difference between the 75th and 25thpercentile is called the interquartile range and a marker to quantifyscattering in the population. All constructs had only one singlecassette insertion in the soybean genome.

In Table 5, all exemplary HPPD polypeptide variants are better in allpercentile analyses than the prior art mutant HPPD polypeptide(WO2014/043435) corresponding to SEQ ID NO:2 in this invention. Thescoring of the prior art mutant HPPD polypeptide (WO2014/043435)corresponding to SEQ ID NO:2 in this invention is listed in the bottomrow of Table 5.

TABLE 5 Evaluation of leaf area damage from transgenic soybean T0 eventsfive days after the application of Cmpd. 1 at a rate of 300 g AI/ha bypercentile distribution. Total T0 Soybean number Events independentInter- expressing events quartile polpypetide of sprayed 25th Median75th range 90th SEQ ID NO: 47 57 5.0 15.0 15.0 10.0 51.0 SEQ ID NO: 6611 5.0 10.0 25.0 20.0 41.0 SEQ ID NO: 68 14 5.0 10.0 16.3 11.3 20.0 SEQID NO: 69 11 20.0 20.0 25.0 5.0 42.0 SEQ ID NO: 70 9 10.0 15.0 25.0 15.035.0 SEQ ID NO: 2 75 15.0 20.0 45.0 30.0 87.0

Example 6: Cotton T0 Plant Establishment and Selection

Cotton transformation is achieved by using methods well known in theart, especially preferred method in the one described in the PCT patentpublication WO 00/71733. Regenerated plants are transferred to thegreenhouse. Following an acclimation period, sufficiently grown plantsare sprayed with HPPD inhibitor herbicides as for example tembotrioneequivalent to 100 or 200 gAI/ha supplemented with ammonium sulfate andmethyl ester rapeseed oil. Seven days after the spray application, thesymptoms due to the treatment with the HPPD inhibitor herbicide areevaluated and compared to the symptoms observed on wild-type cottonplants subjected to the same treatment under the same conditions.

Example 7: Transformation of Maize Plant Cells by Agrobacterium-MediatedTransformation

Constructing the plant expression cassette for stable expression in themaize plant and maize transformation are well known in the art and inthis particular example the methods were described and used fromWO2014/043435 and WO2008/100353. The polynucleotide sequences encodingthe mutant HPPD polypeptides in this application are stacked with apolynucleotide sequence encoding an EPSPS protein variant to confertolerance to herbicides, which target the EPSPS. The EPSPS gene wasisolated and mutated from Arthrobacter globiformis (WO2008/100353) andjoined in-frame to a transit peptide sequence to guide translocation ofthe translated protein to the chloroplast. Stable expression is achievedwith a ubiquitous promoter (Ubiquitin 4 promoter from sugarcane, U.S.Pat. No. 6,638,766), and a 35S terminator sequence from CauliflowerMosaic Virus, which is cloned upstream and downstream of the EPSPS gene,respectively.

The corresponding mutant HPPD polypeptide will be cloned with the samepromoter, chloroplast transit peptide, and terminator sequence asdescribed for the EPSPS gene expression cassette. The coding sequencesfor both genes are codon optimized for maize expression. For the maizetransformation ears are best collected 8-12 days after pollination.Embryos are isolated from the ears, and those embryos 0.8-1.5 mm in sizeare preferred for use in transformation. Embryos are plated scutellumside-up on a suitable incubation media, and incubated overnight at 25°C. in the dark.

However, it is not necessary per se to incubate the embryos overnight.Embryos are contacted with an Agrobacterium strain containing theappropriate vectors having a nucleotide sequence of the presentinvention for Ti plasmid mediated transfer for about 5-10 min, and thenplated onto co-cultivation media for about 3 days (25° C. in the dark).After co-cultivation, explants are transferred to recovery period mediafor about five days (at 25° C. in the dark). Explants are incubated inselection media with glyphosate for up to eight weeks, depending on thenature and characteristics of the particular selection utilized. Afterthe selection period, the resulting callus is transferred to embryomaturation media, until the formation of mature somatic embryos isobserved. The resulting mature somatic embryos are then placed under lowlight, and the process of regeneration is initiated as known in the art.The resulting shoots are allowed to root on rooting media, and theresulting plants are transferred to nursery pots and propagated astransgenic plants. Plants are routinely analyzed for the expression andpresence of the transgenes using the ELISA protein detection method.Only plants recovering in the selection media and having a detectableHPPD transgene protein expression are used for the herbicide toleranceanalysis.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. A recombinant nucleic acid molecule encoding an enzymatically active4-hydroxyphenylpyruvate dioxygenase (HPPD) polypeptide whose amino acidsequence (1) comprises the substitutions (a) an alanine at the aminoacid position corresponding to amino acid position 268 of SEQ ID NO:1,(b) a proline at the amino acid position corresponding to amino acidposition 335 of SEQ ID NO:1, (c) a histidine or an aspartic acid at theposition corresponding to amino acid position 336 of SEQ ID NO:1, and(d) a serine at the position corresponding to amino acid position 337 ofSEQ ID NO:1; or (II) comprises the substitutions of (I) and furthercomprises i. a lysine or leucine at the amino acid positioncorresponding to amino acid position 213 of SEQ ID NO:1; and/or ii. analanine at the amino acid position corresponding to amino acid position215 of SEQ ID NO:1; and/or iii. an arginine, asparagine, leucine,glutamic acid, proline or serine at the amino acid positioncorresponding to amino acid position 270 of SEQ ID NO:1; and/or iv. anarginine, lysine, glutamine, methionine or histidine at the amino acidposition corresponding to amino acid position 315 of SEQ ID NO:1; and/orv. an arginine, glutamine, methionine, glutamic acid, glycine, leucine,or valine at the amino acid position corresponding to amino acidposition 340 of SEQ ID NO:1; and/or vi. a glutamine, proline, orarginine at the amino acid position corresponding to amino acid position344 of SEQ ID NO:1; and/or vii. a lysine, arginine, methionine, alanine,or valine at the amino acid position corresponding to amino acidposition 345 of SEQ ID NO:1; or (III) comprises the substitutions of (I)and further comprises i. a leucine or lysine at the amino acid positioncorresponding to amino acid position 213 of SEQ ID NO:1; and/or ii. analanine at the amino acid position corresponding to amino acid position215 of SEQ ID NO:1; and/or iii. an asparagine, glutamic acid or serineat the amino acid position corresponding to amino acid position 270 ofSEQ ID NO:1; and/or iv. an arginine, lysine, glutamine or methionine atthe amino acid position corresponding to amino acid position 315 of SEQID NO:1; and/or v. an arginine, or valine at the amino acid positioncorresponding to amino acid position 340 of SEQ ID NO: 1; and/or vi. aglutamine at the amino acid position corresponding to amino acidposition 344 of SEQ ID NO:1; and/or vii. a lysine, valine, or methionineat the amino acid position corresponding to amino acid position 345 ofSEQ ID NO:1; or (IV) comprises the substitutions of (I) and furthercomprises i. a lysine at the amino acid position corresponding to aminoacid position 213 of SEQ ID NO:1; and/or ii. an asparagine, glutamicacid or serine at the amino acid position corresponding to amino acidposition 270 of SEQ ID NO: 1; and/or iii. an arginine, lysine, glutamineat the amino acid position corresponding to amino acid position 315 ofSEQ ID NO:1; and/or iv. an arginine or valine at the amino acid positioncorresponding to amino acid position 340 of SEQ ID NO:1; and/or v. aglutamine at the amino acid position corresponding to amino acidposition 344 of SEQ ID NO:1; and/or vi. a lysine, valine, or methionineat the amino acid position corresponding to amino acid position 345 ofSEQ ID NO:1; or (V) comprises the substitutions of any one of (I)-(IV)and has at least 53% sequence identity to the amino acid sequence setforth in SEQ ID NO: 1; and wherein said HPPD polypeptide is tolerant toone or more HPPD inhibitor herbicide(s).
 2. (canceled)
 3. (canceled) 4.(canceled)
 5. (canceled)
 6. The recombinant nucleic acid molecule ofclaim 1, wherein its nucleotide sequence is a synthetic sequence thathas been designed for expression in a plant.
 7. The recombinant nucleicacid molecule of any of claim 1, wherein its nucleotide sequence isoperably linked to a promoter capable of directing expression of thenucleotide sequence in a plant cell.
 8. The recombinant nucleic acidmolecule of claim 1, wherein said HPPD inhibitor herbicide is selectedfrom the group consisting of triketones, diketonitriles, isoxazoles,hydroxypyrazoles, N-(1,2,5-oxadiazol-3yl)benzamides,N-(1,3,4-oxadiazol-2-yl)benzamides, N-(tetrazol-5-yl)- orN-(triazol-5-yl)arylcarboxamides, pyridazinone derivatives, oxoprazinederivatives, N-(triazol-2-yl)arylcarboxamides, triazinones, pyrazolones.9. The recombinant nucleic acid molecule of claim 8, wherein said HPPDinhibitor herbicide is selected from the group consisting ofbenzobicyclon, sulcotrione, mesotrione, tembotrione, tefuryltrione,bicyclopyrone, fenquinotrione, isoxaflutole, diketonitrile, pyrazoxyfen,benzofenap, pyrazolynate, pyrasulfotole, topramezone, tolpyralate,2-methyl-N-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide,2-chloro-3-ethoxy-4-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide,4-(difluoromethyl)-2-methoxy-3-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide,2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)-benzamide,and2-(methoxymethyl)-3-(methylsulfinyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide.10. A host cell that contains the recombinant nucleic acid molecule ofclaim
 1. 11. The host cell of claim 10 that is a bacterial host cell.12. The host cell of claim 10 that is a plant cell.
 13. A transgenicplant comprising the recombinant nucleic acid molecule of claim
 1. 14.The plant of claim 13, wherein said plant is selected from the groupconsisting of maize, sorghum, wheat, sunflower, tomato, crucifers,peppers, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco,barley, and oilseed rape.
 15. A transgenic seed comprising therecombinant nucleic acid molecule of claim
 1. 16. A recombinantpolypeptide comprising an enzymatically active HPPD polypeptide, whereinsaid HPPD polypeptide is tolerant to one or more HPPD inhibitorherbicide(s) and wherein said HPPD polypeptide (1) comprises thesubstitutions (a) an alanine at the amino acid position corresponding toamino acid position 268 of SEQ ID NO:1, (b) a proline at the amino acidposition corresponding to amino acid position 335 of SEQ ID NO:1, (c) ahistidine or an aspartic acid at the position corresponding to aminoacid position 336 of SEQ ID NO:1, and (d) a serine at the positioncorresponding to amino acid position 337 of SEQ ID NO:1; or (II)comprises the substitutions of (I) and further comprises i. a lysine orleucine at the amino acid position corresponding to amino acid position213 of SEQ ID NO:1; and/or ii. an alanine at the amino acid positioncorresponding to amino acid position 215 of SEQ ID NO:1; and/or iii. anarginine, asparagine, leucine, glutamic acid, proline or serine at theamino acid position corresponding to amino acid position 270 of SEQ IDNO:1; and/or iv. an arginine, lysine, glutamine, methionine or histidineat the amino acid position corresponding to amino acid position 315 ofSEQ ID NO:1; and/or v. an arginine, glutamine, methionine, glutamicacid, glycine, leucine, or valine at the amino acid positioncorresponding to amino acid position 340 of SEQ ID NO:1; and/or vi. aglutamine, proline, or arginine at the amino acid position correspondingto amino acid position 344 of SEQ ID NO: 1; and/or vii. a lysine,arginine, methionine, alanine, or valine at the amino acid positioncorresponding to amino acid position 345 of SEQ ID NO:1; or (III)comprises the substitutions of (I) and further comprises i. a leucine orlysine at the amino acid position corresponding to amino acid position213 of SEQ ID NO:1; and/or ii. an alanine at the amino acid positioncorresponding to amino acid position 215 of SEQ ID NO:1; and/or iii. anasparagine, glutamic acid or serine at the amino acid positioncorresponding to amino acid position 270 of SEQ ID NO: 1; and/or iv. anarginine, lysine, glutamine or methionine at the amino acid positioncorresponding to amino acid position 315 of SEQ ID NO:1; and/or v. avaline at the amino acid position corresponding to amino acid position340 of SEQ ID NO:1; and/or vi. a glutamine at the amino acid positioncorresponding to amino acid position 344 of SEQ ID NO:1; and/or vii. alysine, valine, or methionine at the amino acid position correspondingto amino acid position 345 of SEQ ID NO:1, and wherein said HPPDpolypeptide is tolerant to one or more HPPD inhibitor herbicide(s); or(IV) comprises the substitutions of (I) and further comprises i. alysine at the amino acid position corresponding to amino acid position213 of SEQ ID NO:1; and/or ii. an asparagine, glutamic acid or serine atthe amino acid position corresponding to amino acid position 270 of SEQID NO:1; and/or iii. an arginine, lysine, glutamine at the amino acidposition corresponding to amino acid position 315 of SEQ ID NO:1; and/oriv. a valine at the amino acid position corresponding to amino acidposition 340 of SEQ ID NO:1; and/or v. a glutamine at the amino acidposition corresponding to amino acid position 344 of SEQ ID NO:1; and/orvi. a lysine, valine, or methionine at the amino acid positioncorresponding to amino acid position 345 of SEQ ID NO:1; or (V)comprises the substitutions of (I) and further comprises i. anasparagine, glutamic acid or serine at the amino acid positioncorresponding to amino acid position 270 of SEQ ID NO: 1; and/or ii. anarginine, lysine, glutamine at the amino acid position corresponding toamino acid position 315 of SEQ ID NO:1; and/or iii. a valine at theamino acid position corresponding to amino acid position 340 of SEQ IDNO:1; and/or iv. a glutamine at the amino acid position corresponding toamino acid position 344 of SEQ ID NO:1; and/or v. a lysine, valine, ormethionine at the amino acid position corresponding to amino acidposition 345 of SEQ ID NO:1; or (VI) comprises the substitutions of anyone of (I)-(V) and has at least 53% sequence identity to the amino acidsequence set forth in SEQ ID NO:1.
 17. (canceled)
 18. (canceled) 19.(canceled)
 20. (canceled)
 21. A recombinant polypeptide comprising anHPPD polypeptide, wherein said HPPD polypeptide is tolerant to one ormore HPPD inhibitor herbicide(s) and wherein said HPPD protein comprisesan alanine at the amino acid position corresponding to amino acidposition 268 of SEQ ID NO:1, an asparagine or serine at the amino acidposition corresponding to amino acid position 270 of SEQ ID NO:1, aproline at the amino acid position corresponding to amino acid position335 of SEQ ID NO:1, a histidine or an aspartic acid at the positioncorresponding to amino acid position 336 of SEQ ID NO:1, a serine at theposition corresponding to amino acid position 337 of SEQ ID NO:1, avaline at the amino acid position corresponding to amino acid position340 of SEQ ID NO:1, a glutamine at the amino acid position correspondingto amino acid position 344 of SEQ ID NO:1 and a valine, or methionine,or lysine at the amino acid position corresponding to amino acidposition 345 of SEQ ID NO:1.
 22. (canceled)
 23. The recombinantpolypeptide of claim 16, wherein said HPPD inhibitor herbicide isselected from the group consisting of triketones, diketontriles,isoxazoles, hydroxypyrazoles, N-(1,2,5-oxadiazol-3-yl)benzamides,N-(1,3,4-oxadiazol-2-yl)benzamides, N-(tetrazol-5-yl)- orN-(triazol-5-yl)arylcarboxamides, pyridazinone derivatives, oxoprazinederivatives, N-(triazol-2-yl)arylcarboxamides, triazinones, andpyrazolones.
 24. The recombinant polypeptide of claim 23, wherein saidHPPD inhibitor herbicide is selected from the group consisting ofbenzobicyclon, sulcotrione, mesotrione, tembotrione, tefuryltrione,bicyclopyrone, fenquinotrione, isoxaflutole, diketonitrile pyrazoxyfen,benzofenap, pyrazolynate, pyrasulfotole, topramezone, tolpyralate,2-methyl-N-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide,2-chloro-3-ethoxy-4-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide,4-(difluoromethyl)-2-methoxy-3-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide,2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)-benzamide,an2-(methoxymethyl)-3-(methylsulfinyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide.25. A method for producing a polypeptide with HPPD inhibitor herbicidetolerance activity, comprising culturing the host cell of claim 10 underconditions in which a nucleic acid molecule encoding the polypeptide isexpressed.
 26. The plant of claim 13, wherein said plant has stablyincorporated into its genome a DNA construct, said construct comprisinga promoter operably linked with the nucleic acid encoding said HPPDpolypeptide.
 27. A plant cell, a plant tissue, or a plant seed of aplant of claim
 26. 28. The plant of claim 26, wherein said plant isselected from the group consisting of maize, sorghum, wheat, sunflower,tomato, crucifers, peppers, potato, cotton, rice, soybean, sugar beet,sugarcane, tobacco, barley, and oilseed rape.
 29. Transgenic seed of theplant of claim 26, having said DNA construct stably incorporated in itsgenome.
 30. A method of controlling weeds in a field comprising plantingthe plant of claim 26 or a seed thereof in a field and applying to saidfield an effective concentration of an HPPD inhibitor herbicide.
 31. Themethod of claim 30, wherein said HPPD inhibitor herbicide is selectedfrom the group consisting of triketones, diketonitriles, isoxazoles,hydroxypyrazoles, N-(1,2,5-oxadiazol-3-yl)benzamides,N-(1,3,4-oxadiazol-2-yl)benzamides, N-(tetrazol-5-yl)- orN-(triazol-5-yl)arylcarboxamides, pyridazinone derivatives, oxoprazinederivatives, N-(triazol-2-yl)arylcarboxamides, triazinones, andpyrazolones.
 32. The method of claim 31, wherein said HPPD inhibitorherbicide is selected from the group consisting of benzobicyclon,sulcotrione, mesotrione, tembotrione, tefuryitrione, bicyclopyrone,fenquinotrione, isoxaflutole, diketonitrile pyrazoxyfen, benzofenap,pyrazolynate, pyrasulfotole, topramezone, tolpyralate,2-methyl-N-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide,2-chloro-3-ethoxy-4-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide,4-(difluoromethyl)-2-methoxy-3-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide,2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide,an2-(methoxymethyl)-3-(methylsulfinyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide.33. Use of the nucleic acid of claim 1 for rendering a plant tolerant toone or more HPPD inhibitor herbicide(s).
 34. A commodity productcomprising an HPPD polypeptide having the HPPD amino acid sequence ofclaim 16, or comprising the nucleic acid molecule encoding saidpolypeptide, wherein said product is selected from the group consistingof whole or processed seeds or grain, animal feed, corn or soybean meal,corn or soybean flour, corn starch, soybean meal, soybean flakes,soybean protein concentrate, soybean protein isolates, texturizedsoybean protein concentrate, cosmetics, hair care products, soybean nutbutter, natto, tempeh, hydrolyzed soybean protein, whipped topping,shortening, lecithin, edible whole soybeans, soybean yogurt, soybeancheese, tofu, yuba, and cooked, polished, steamed, baked or parboiledgrain.